MicrobiologyBytes

As a result of its high mutation rate, human immunodeficiency virus (HIV) can evade recognition by the host immune response through the generation of virus variants, so-called “escape mutants”. This avoidance of cytotoxic T lymphocyte (CTL) mediated killing seems to be one of the major reasons why virus replication is not controlled effectively. However, it has been difficult to investigate the dynamics of immune escape.

Researchers from Utrecht University in The Netherlands have developed a new mathematical model that illustrates how HIV evades the immune system. The computational model of HIV infection consists of several CTL clones that can recognize specific parts of virus proteins called “epitopes”. The simulation makes it possible follow the dynamics of immune escape in detail and helps to interpret longitudinal data of real HIV infections by modeling detailed interactions between a mutating virus and the immune system. HIV avoids recognition by the human immune response through the generation of virus variants called “escape mutants”. This prevents effective control of virus replication, eventually causing HIV-infected patients to progress to AIDS. However, it remains difficult to fully understand the dynamics of immune escape, as data from infected patients is relatively sparse. The new work uses computer simulations to help interpret longitudinal data derived from HIV-infected patients. These illustrate that the virus usually evades the immune response very slowly, on a timescale lasting years. Depending on the diversity of the host immune system, the virus will either be controlled effectively or accumulate detrimental mutations.

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Interestingly, changing the relative sizes of the CTL clones leads to a different evolution path for the virus. Instead of reducing the number of infected cells, an alternative strategy of vaccine design could be to reduce the replicative capacity of the virus by imposing a fitness cost in virus replication or infectivity. That might have important implications for disease progression.

Dynamics of Immune Escape during HIV/SIV Infection. 2008 PLoS Comput Biol 4(7): e1000103
Several studies have shown that cytotoxic T lymphocytes (CTLs) play an important role in controlling HIV/SIV infection. Notably, the observation of escape mutants suggests a selective pressure induced by the CTL response. However, it remains difficult to assess the definite role of the cellular immune response. We devise a computational model of HIV/SIV infection having a broad cellular immune response targeting different viral epitopes. The CTL clones are stimulated by viral antigen and interact with the virus population through cytotoxic killing of infected cells. Consequently, the virus population reacts through the acquisition of CTL escape mutations. Our model provides realistic virus dynamics and describes several experimental observations. We postulate that inter-clonal competition and immunodominance may be critical factors determining the sequential emergence of escapes. We show that even though the total killing induced by the CTL response can be high, escape rates against a single CTL clone are often slow and difficult to estimate from infrequent sequence measurements. Finally, our simulations show that a higher degree of immunodominance leads to more frequent escape with a reduced control of viral replication but a substantially impaired replicative capacity of the virus. This result suggests two strategies for vaccine design: Vaccines inducing a broad CTL response should decrease the viral load, whereas vaccines stimulating a narrow but dominant CTL response are likely to induce escape but may dramatically reduce the replicative capacity of the virus.

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Swiss pharmaceutical company Roche will suspend its HIV research because none of its pending medicines represent significant improvement over existing drugs. Company scientists currently working in HIV will be reassigned to other activities.

Reuters

The public health approach to HIV treatment, in which a limited number of drug combinations is used for all patients in South African programs, works just as well as the highly individualized approach to drug selection used in Switzerland. Researchers based at University of Bern, Switzerland and University of Cape Town, South Africa, analyzed data collected since 2001 from more than 2,000 patients enrolled in HIV treatment programs in two townships (Gugulethu and Khayelitsha) in Cape Town, South Africa, and from more than 1,000 patients enrolled in the Swiss HIV Cohort Study, a nationwide study of HIV-infected people. Currently, approximately 3 million people with HIV in low- and middle-income countries are receiving antiretroviral therapy. The majority of treated individuals are in sub-Saharan Africa, where only about 50,000 people were being treated as recently as 5 years ago. This rapid scale-up has raised questions of whether the World Health Organization s standardized approach to treatment selection and clinical monitoring can meet with the same success as customized approaches used in high-income countries. This study found the programmatic and individualized approaches to be equally successful provided that treatment is begun early enough. The patients in South Africa started their treatment for HIV infection with one of four first-line therapies, and about a quarter changed to a second-line therapy during the study. By contrast, 36 first-line regimens were used in Switzerland, where half the patients changed to a different regimen. Despite these differences, the level of HIV in blood was greatly reduced within a year in virtually all the patients and viral rebound (an increase in viral levels following initially effective treatment) developed within 2 years in a quarter of the patients in both countries. However, more patients died in South Africa than in Switzerland, particularly during the first 3 months of therapy. According to the researchers, this difference likely reflects the fact that patients in South Africa were more likely than patients in Switzerland to have advanced AIDS prior to starting treatment. These findings support the continued rollout of the public-health approach to HIV treatment in resource-poor countries, and raise the possibility that a more standardized approach could be taken in developed countries without compromising treatment effectiveness. The higher mortality in South Africa compared to Switzerland suggests that many HIV-infected patients in resource-limited countries would benefit from earlier initiation of therapy.

Public-health and individual approaches to antiretroviral therapy: Township South Africa and Switzerland compared. 2008 PLoS Med 5(7): e148. doi:10.1371/journal.pmed.0050148
Acquired immunodeficiency syndrome (AIDS) has killed more than 25 million people since the first reported case in 1981, and more than 30 million people 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-infected people died within 10 years of becoming infected. Then, in 1996, highly active antiretroviral therapy (HAART) – a combination of several antiretroviral drugs – was developed. Now, in resource-rich countries, clinicians provide individually tailored care for HIV-infected people by prescribing combinations of antiretroviral drugs chosen from more than 20 approved medicines. The approach to treatment of HIV in developed countries typically also includes frequent monitoring of the amount of virus in patients’ blood (viral load), viral resistance testing (to see whether any viruses are resistant to specific antiretroviral drugs), and regular CD4 cell counts (an indication of immune-system health). Since the implementation of these interventions, the health and life expectancy of people with HIV has improved dramatically in these countries.

Why Was This Study Done?
The history of HIV care in resource-poor countries has been very different. Initially, these countries could not afford to provide HAART for their populations. In 2003, however, governments, international agencies, and funding bodies began to implement plans to increase HAART coverage in developing countries. By December 2006, more than a quarter of the HIV-infected people in low- and middle-income countries who urgently needed treatment were receiving HAART. However, instead of individualized treatment, HAART programs in developing countries follow a public-health approach developed by the World Health Organization. That is, drug regimens, clinical decision-making, and clinical and laboratory monitoring are all standardized. This public-health approach takes into account the realities of under-resourced health systems, but is it as effective as the individualized approach? The researchers addressed this question by comparing virologic responses (the effect of treatment on the viral load), changes to first-line (initial) therapy, and deaths in patients receiving HAART in South Africa (public-health approach) and in Switzerland (individualized approach).

What Did the Researchers Do and Find?
The researchers analyzed data collected since 2001 from more than 2,000 patients enrolled in HAART programs in two townships (Gugulethu and Khayelitsha) in Cape Town, South Africa, and from more than 1,000 patients enrolled in the Swiss HIV Cohort Study, a nationwide study of HIV-infected people. The patients in South Africa, who had a lower starting CD4 cell count and were more likely to have advanced AIDS than the patients in Switzerland, started their treatment for HIV infection with one of four first-line therapies, and about a quarter changed to a second-line therapy during the study. By contrast, 36 first-line regimens were used in Switzerland and half the patients changed to a different regimen. Despite these differences, the viral load was greatly reduced within a year in virtually all the patients and viral rebound (an increased viral load after a low measurement) developed within 2 years in a quarter of the patients in both countries. However, more patients died in South Africa than in Switzerland, particularly during the first 3 months of therapy.

What Do These Findings Mean?
These findings suggest that the public health approach to HAART practiced in South Africa is as effective in terms of virologic outcomes as the individualized approach practiced in Switzerland. This is reassuring because it suggests that ‘‘antiretroviral anarchy’’ (the unregulated use of antiretroviral drugs, interruptions in drug supplies, and the lack of treatment monitoring), which is likely to lead to the emergence of viral resistance, is not happening in South Africa as some experts feared it might. Thus, these findings support the continued rollout of the public-health approach to HAART in resource-poor countries. Conversely, they also suggest that a more standardized approach to HAART could be taken in Switzerland (and in other industrialized countries) without compromising its effectiveness. Finally, the higher mortality in South Africa than in Switzerland, which partly reflects the many patients in South Africa in desperate need of HAART and their more advanced disease at the start of therapy, suggests that HIV-infected patients in South Africa and in other resource-limited countries would benefit from earlier initiation of therapy.

Cross-species transmissions of infectious agents from primates to humans have led to major disease outbreaks, with AIDS representing a particularly serious example. It has recently been shown that humans who hunt primates frequently acquire simian foamy virus (SFV) infections. These viruses have been proposed as an “early warning system” of human exposure to wild primates. In a recent study, researchers in Cameroon, the Democratic Republic of Congo, France, Gabon, Germany, Japan, Rwanda, the United Kingdom, and the United States found that simian foamy virus (SFV) is widespread among wild chimpanzees throughout equatorial Africa using novel non-invasive methods. They analyzed more than 700 fecal samples from 25 chimpanzee communities across sub-Saharan Africa and obtained viral sequences from a large number of these. SFV was widespread among all chimpanzee subspecies, with infection rates ranging from 44% to 100%. The new viruses formed subspecies-specific lineages consistent with virus/host co-evolution. We also found mosaic sequences due to recombination, indicating that chimpanzees can be infected with multiple viral strains. One chimpanzee harbored an SFV from a monkey species, indicating cross-species transmission in the wild. These data indicate that chimpanzees represent a substantial natural reservoir of SFV. Monitoring humans for these viruses should identify locations where human/chimpanzee encounters are most frequent, and where additional transmissions of chimpanzee pathogens should be anticipated. Identifying the locations may help to determine where the highest rates of human–chimpanzee interactions occur, and may predict what other pathogens may jump the species barrier next.

Molecular Ecology and Natural History of Simian Foamy Virus Infection in Wild-Living Chimpanzees. 2008 PLoS Pathog 4(7): e1000097
Identifying microbial pathogens with zoonotic potential in wild-living primates can be important to human health, as evidenced by human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2) and Ebola virus. Simian foamy viruses (SFVs) are ancient retroviruses that infect Old and New World monkeys and apes. Although not known to cause disease, these viruses are of public health interest because they have the potential to infect humans and thus provide a more general indication of zoonotic exposure risks. Surprisingly, no information exists concerning the prevalence, geographic distribution, and genetic diversity of SFVs in wild-living monkeys and apes. Here, we report the first comprehensive survey of SFVcpz infection in free-ranging chimpanzees (Pan troglodytes) using newly developed, fecal-based assays. Chimpanzee fecal samples were collected at 25 field sites throughout equatorial Africa and tested for SFVcpz-specific antibodies or viral nucleic acids. SFVcpz infection was documented at all field sites, with prevalence rates ranging from 44% to 100%. In two habituated communities, adult chimpanzees had significantly higher SFVcpz infection rates than infants and juveniles, indicating predominantly horizontal rather than vertical transmission routes. Some chimpanzees were co-infected with simian immunodeficiency virus (SIVcpz); however, there was no evidence that SFVcpz and SIVcpz were epidemiologically linked. SFVcpz nucleic acids were recovered from 177 fecal samples, all of which contained SFVcpz RNA and not DNA. Phylogenetic analysis of partial gag, pol-RT and pol-IN sequences identified a diverse group of viruses, which could be subdivided into four distinct SFVcpz lineages according to their chimpanzee subspecies of origin. Within these lineages, there was evidence of frequent superinfection and viral recombination. One chimpanzee was infected by a foamy virus from a Cercopithecus monkey species, indicating cross-species transmission of SFVs in the wild. These data indicate that SFVcpz (i) is widely distributed among all chimpanzee subspecies; (ii) is shed in fecal samples as viral RNA; (iii) is transmitted predominantly by horizontal routes; (iv) is prone to superinfection and recombination; (v) has co-evolved with its natural host; and (vi) represents a sensitive marker of population structure that may be useful for chimpanzee taxonomy and conservation strategies.

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With two catalytic activities and many substrates, how does HIV’s reverse transcriptase enzyme know what to do to which substrate? Zooming in on the enzyme’s molecular interactions provides tantalizing clues.

Dynamic binding orientations direct activity of HIV reverse transcriptase. Nature 453, 184-189
The reverse transcriptase of human immunodeficiency virus (HIV) catalyses a series of reactions to convert the single-stranded RNA genome of HIV into double-stranded DNA for host-cell integration. This task requires the reverse transcriptase to discriminate a variety of nucleic-acid substrates such that active sites of the enzyme are correctly positioned to support one of three catalytic functions: RNA-directed DNA synthesis, DNA-directed DNA synthesis and DNA-directed RNA hydrolysis. However, the mechanism by which substrates regulate reverse transcriptase activities remains unclear. Here we report distinct orientational dynamics of reverse transcriptase observed on different substrates with a single-molecule assay. The enzyme adopted opposite binding orientations on duplexes containing DNA or RNA primers, directing its DNA synthesis or RNA hydrolysis activity, respectively. On duplexes containing the unique polypurine RNA primers for plus-strand DNA synthesis, the enzyme can rapidly switch between the two orientations. The switching kinetics were regulated by cognate nucleotides and non-nucleoside reverse transcriptase inhibitors, a major class of anti-HIV drugs. These results indicate that the activities of reverse transcriptase are determined by its binding orientation on substrates.

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• The shape of HIV RNA
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Viruses are small infectious agents responsible for many human diseases, including acquired immunodeficiency syndrome (AIDS). Like other viruses, the human immunodeficiency virus 1 (HIV-1; the cause of AIDS) enters human cells and uses the cellular machinery to replicate before bursting out of its temporary home. During the initial stage of HIV infection, a particular group of cells in the human immune system, CD8⁺ T cells, are thought to be important in controlling the level of the virus. These immune system cells recognize pieces of viral protein called antigens displayed on the surface of infected cells; different subsets of CD8⁺ T cells recognize different antigens. When a CD8⁺ T cell recognizes its specific antigen (or more accurately, a small part of the antigen called an epitope ), it releases cytotoxins (which kill the infected cells) and cytokines, proteins that stimulate CD8⁺ T cell proliferation and activate other parts of the immune system. With many viruses, when a person first becomes infected (an acute viral infection), antigen-specific CD8⁺ T cells completely clear the infection. But with HIV-1 and some other viruses, these cells do not manage to remove all the viruses from the body and a chronic (long-term) infection develops, during which the immune system is constantly exposed to viral antigen.

In HIV-1 infections (and other chronic viral infections), virus-specific CD8⁺ T cells lose their ability to proliferate, to make cytokines, and to kill infected cells as patients progress to the longterm stages of infection. That is, the virus-specific CD8⁺ T cells gradually lose their effector functions and become functionally impaired or exhausted. Polyfunctional CD8⁺ T cells (those that release multiple cytokines in response to antigen) are believed to be essential for an effective CD8⁺ T cell response, so scientists trying to develop HIV-1 vaccines would like to stimulate the production of this type of cell. To do this they need to understand why these polyfunctional cells are lost during chronic infections. Is their loss the cause or the result of viral persistence? In other words, does the constant presence of viral antigen lead to the exhaustion of CD8⁺ T cells during chronic HIV infection? In this study, the researchers investigate this question by looking at the polyfunctionality of CD8⁺ cells responding to several different viral epitopes at various times during HIV-1 infection, starting very early after infection with HIV-1 had occurred.

The researchers enrolled 18 patients recently infected with HIV-1 and analyzed their CD8⁺ T cell responses to specific epitopes at various times after enrollment using a technique called flow cytometry. They found that the epitope-specific CD8⁺ cells produced several effector proteins after antigen stimulation during the initial stage of HIV-1 infection, but lost their polyfunctionality in the face of persistent viral infection. The CD8⁺ T cells also increased their production of programmed death 1 (PD-1), a protein that has been shown to be associated with the functional impairment of CD8⁺ T cells. Some of the patients began antiretroviral therapy during the study, and the researchers found that this treatment, which reduced the viral load, reversed CD8⁺ T cell exhaustion. Finally, the appearance in the patients blood of viruses that had made changes in the specific epitopes recognized by the CD8⁺ T cells to avoid being killed by these cells, also reversed the exhaustion of the T cells recognizing these particular epitopes.

These findings suggest that the constant presence of HIV-1 antigen causes the functional impairment of virus-specific CD8⁺ T cell responses during chronic HIV-1 infections. Treatment with antiretroviral drugs reversed this functional impairment by reducing the amount of antigen in the patients. Similarly, the appearance of viruses with altered epitopes, which effectively reduced the amount of antigen recognized by those epitope-specific CD8⁺ T cells without reducing the viral load, also reversed T cell exhaustion. These results would not have been seen if the functional impairment of CD8⁺ cells were the cause rather than the result of antigen persistence. By providing new insights into how the T cell response to viruses evolves during persistent viral infections, these findings should help in the design of vaccines against HIV and other viruses that cause chronic viral infections.

Antigen load and viral sequence diversification determine the functional profile of HIV-1 specific CD8 T cells. PLoS Med 5(5): e100

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The function of the RNA genome of the human immunodeficiency virus (HIV) is determined both by its sequence and by its ability to fold back on itself to form specific higher-order structures. In order to describe physical structures in a region of the HIV RNA genome known to play multiple, critical roles in viral replication and pathogenesis. In this week’s PLoS Biology scientists from the University of North Carolina show how they have devised a high-throughput, quantitative, and comprehensive structure-mapping approach that locates flexible (unpaired) nucleotides within a folded RNA, assaying hundreds of nucleotides at a time. They find that the first 10% of the HIV-1 genome has a single predominant structure and that regulatory motifs have significantly greater structure than do protein-coding segments. The HIV genome interacts with numerous proteins, including multiple copies of nucleocapsids. They also directly map RNA-protein interactions inside virions and discover that the nucleocapsid interacts with viral RNA in at least three distinct ways, depending on the context within the overall genome structure. The group hopes that further application of the high-throughput RNA-structure analysis tools described will make it possible to address diverse structure-function relationships in intact cellular and viral RNAs.

Replication and pathogenesis of the human immunodeficiency virus (HIV) is tightly linked to the structure of its RNA genome, but genome structure in infectious virions is poorly understood. High-throughput SHAPE (selective 29-hydroxyl acylation analyzed by primer extension) technology uses many of the same tools as DNA sequencing, was used to quantify RNA backbone flexibility at single-nucleotide resolution and from which robust structural information can be immediately derived. We analyze the structure of HIV-1 genomic RNA in four biologically instructive states, including the authentic viral genome inside native particles. Remarkably, given the large number of plausible local structures, the first 10% of the HIV-1 genome exists in a single, predominant conformation in all four states. We also discover that noncoding regions functioning in a regulatory role have significantly lower SHAPE reactivities, and hence more structure, than do viral coding regions that function as the template for protein synthesis. By directly monitoring protein binding inside virions, we identify the RNA recognition motif for the viral nucleocapsid protein. Seven structurally homologous binding sites occur in a well-defined domain in the genome, consistent with a role in directing specific packaging of genomic RNA into nascent virions. In addition, we identify two distinct motifs that are targets for the duplex destabilizing activity of this same protein. The nucleocapsid protein destabilizes local HIV-1 RNA structure in ways likely to facilitate initial movement both of the retroviral reverse transcriptase from its tRNA primer and of the ribosome in coding regions. Each of the three nucleocapsid interaction motifs falls in a specific genome domain, indicating that local protein interactions can be organized by the long-range architecture of an RNA. High-throughput SHAPE reveals a comprehensive view of HIV-1 RNA genome structure, and further application of this technology will make possible newly informative analysis of any RNA in a cellular transcriptome.
High-throughput SHAPE analysis reveals structures in HIV-1 genomic RNA strongly conserved across distinct biological states. 2008 PLoS Biol 6: e96

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The structure of all retroviruses is similar, although there are some minor differences. Virus particles are far too small to see, the closest we can come to are electron micrographs. To make transmission electron micrographs, the specimen (containing virus particles) are fixed and stained with a metal-containing dye. The more dye different areas of the specimen take up, the darker they appear in the electron micrograph.

In the centre of an HIV particle, there are two molecules of RNA which together make up the genome of the virus. Associated with the RNA are two enzymes, reverse transcriptase and integrase. The genome is enclosed in a conical core consisting of the nucleocapsid proteins. Outside this is an icosahedral protein capsid, which in turn is enclosed by the matrix protein layer. The whole particle is surrounded by a lipid bilayer known as the virus envelope. The transmembrane protein penetrates through the envelope and anchors the surface glycoprotein on the outside of the particle.

To see more detail in virus particles, special imaging techniques are needed. Cryo-electron tomography makes a three dimensional reconstruction from a series of two dimensional transmission electron micrographs taken at extremely low temperatures in order to preserve the structure of the particle. The individual micrographs represent slices though the virus particle which are put together on a computer to construct a three dimensional representation with false colours added for additional clarity.

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In the absence of interventions, 30-45% of exposed infants acquire human immunodeficiency virus type 1 (HIV-1) through mother-to-child transmission. It remains unclear why some infants become infected while others do not, despite significant exposure to HIV-1 in utero, during delivery and while breastfeeding. Ths paper discusses the correlates of vertical transmission with an emphasis on factors that increase maternal HIV-1 levels, either systemically or locally in genital secretions and breast milk. Immune responses may influence maternal viral load, and data suggest that maternal neutralising antibodies reduce infection rates. In addition, infants may be capable of mounting HIV-specific cellular immune responses. The authors propose that both humoral and cellular responses are necessary to reduce infection because cell-free as well as cell-associated virus appears to play a role in vertical transmission. These distinct forms of the virus may be targeted most effectively by different components of the immune system. They also discuss the use of antiretrovirals to reduce transmission, focusing on the mechanisms of action of regimens currently used in developing country settings. Their conclusion is that that prevention relies not only on reducing maternal HIV-1 levels within blood, genital tract and breast milk, but also on pre- and/or post-exposure prophylaxis to the infant. However, HIV-1 has the capacity to mutate under drug pressure and rapidly acquires mutations conferring antiretroviral resistance. This review concludes with data on persistence of low-level resistance after delivery as well as recent guidelines for maternal and infant regimens designed to limit resistance.

Biological mechanisms of vertical human immunodeficiency virus (HIV-1) transmission. 2007 Rev Med Virol. 17: 381-403

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