Combined Immunotherapy as a means of stopping prolonged treatment with ART?

The HIV-1 virus has evolved to exploit the human immune system and counteract all immune defenses mounted against it.  Many different vaccination approaches and therapy treatments have been attempted to control, eliminate, or prevent HIV-1 infection, however, these attempts have not proved successful for virus elimination from the body. 

One approach that has been used to combat HIV-1 infection is the use of ART, or antiretroviral therapy, and this approach has proven to be effective at reducing viral levels in the blood.  ART suppresses HIV-1 viremia to undetectable levels within 12-48 weeks in a majority of patients.  This therapy is a “cocktail” or combination therapy of three or more drugs, including 2  nucleoside-based reverse transcriptase inhibitors (prevents production of the virus genome by inhibiting RNAàDNA transcription), and one or more of the following drugs: non-nucleoside reverse transcriptase inhibitors, membrane fusion inhibitors, viral protease inhibitors, or integrase inhibitors.  Each of these drugs targets a specific part of the viral lifecycle, making it unable to replicate and infect other cells.  Because each drug is mutually exclusive and targets a different area of the lifecycle, it is unlikely that a virus will develop resistance to all three drugs.  Therefore, because the virus does not produce escape mutants from the therapy, this approach is effective at reducing viral levels in the blood (ART information) (more on ART) However, by the time ART is put into effect, the virus has already established residence in T cells in the body, and can continue to infect other cells undetected by the drug by avoiding exposure in the blood via direct cell-to-cell infection.  Another problem with the use of ART for HIV-1 treatment is that it must be administered indefinitely, and if it is discontinued, there is a rapid rebound of viremia, or viral infection of the blood.  Taking ART indefinitely is undesired due to negative side effects (common ART side effects) and resistance to the drugs that develops overtime due to mutation.

Another approach to combat HIV-1 infection has been the use of monoclonal neutralizing anti-HIV-1 antibodies, however, in the past, these antibodies have proven to be ineffective at controlling infection of humanized mice (hu-mice) (Hu-mice as the murine model for the analysis of human hematolymphoid differentiation and function).  However, as of late, more potent antibodies have been uncovered that have decreased viremia in hu-mice and have longer half lives.  Antibodies that target gp120 glycoprotein in the HIV viral envelope have been studied as a potential means of neutralizing HIV infection.  Gp120 binds to CD4 on T cells, enabling initial viral-cell contact before membrane fusion and insertion of the viral genome (more on gp120). 

In a recent study conducted by Horwitz et al (2013) (published in the Proceedings of the National Academy of Sciences of the USA: HIV-1 Suppression and durable control by combining single broadling neutralizing antibodies (bNAbs) and antiretroviral drugs in humanized mice), three antibodies targeting different epitopes of gp120 were used in combination with ART and the effects were analyzed.  The three antibdies were 3BNC117 (a potent CD4 binding site antibody with a long half life), PG16 (which recognizes the V1/V2 loop region), and 10-1074 (which targets the base of the V3 stem). 

New Possible HIV Treatment reduces HIV-1 Infectivity with the use of GANP to mediate APOBEC3G Packaging in HIV-1 Virions

The road to effective HIV vaccines is littered with many hurdles and blockades that cause vaccines to be ineffective. One of the main reasons why HIV leads to many ineffective drug vaccines is because of its ability to mutate rapidly. The rapid mutation of HIV allows the virus to avoid potential vaccines such as antibody vaccines that could neutralize the virus or help facilitate the death of the virus. As a result of the rapid mutation, many current scientists are looking into new ways to fight off the virus. A couple of the new methods in stopping the virus include targeting specific proteins that make up HIV that allow it to mutate and infect immune cells. Scientists are also looking into methods that enhance the host antiviral proteins found in the immune cells. In a new study from Kumamoto University, researchers have discovered a new method in reducing the infectivity of HIV-1 (the usual HIV strain found in humans). What the researchers discovered could lead to a potentially new development of vaccines in fighting HIV. The scientists discovered the use of a recent protein called GANP (germinal center-associated nuclear protein) that enhances the facilitation of the host antiretroviral protein called A3G (its function causes hypermutations in newly synthesized viral DNA) into the viral capsid in the absence of the HIV accessory protein Vif.

The function of HIV’s accessory protein Vif is to promote the synthesis of proviral DNA by inhibiting a host antiviral protein. The host antiviral protein that Vif inhibits is APOBEC3G where Vif degrades the protein by facilitating the APOBEC3G to a proteasome. In the absence of HIV, APOBEC3G normally functions as an antiviral cytidine deaminase enzyme that converts a cytidine to a uracil (C to U) in viral RNA.

In trying to get a visual understanding of the importance of APOBEC3G, here is a video that will demonstrate the general function of APOBEC3G on HIV:

In an effort to take advantage of APOBEC3G’s ability against viral RNA, the researchers tried to experiment and explore the combination of A3G (a protein of the family of APOBEC proteins that has the same ability) with GANP since GANP has shown in a previous study that it enhances the use of one of the other APOBEC proteins called AID. GANP is a protein that is upregulated when CD4⁺ T cells are activated so the researchers want to see how GANP interacts with A3G in the HIV virion during an HIV infection.  

New Mechanism Found That Adds Another Vector For Anti-Retroviral Therapy!

HIV-1 is one of the most well known viruses in the world. The difficulty in combating infection is the rather prevalent mutagenic nature of the virus, which in turn means that anti-retrovirals are quickly rendered inactive after these mutations occur. In addition to this mutagenic nature, HIV-1 has a wide array of accessory proteins that further aids in successful infection. In a recent study by the Journal of Biological Chemistry, researchers focused on one of these accessory HIV-1 proteins, the curious viral protein, Viral Infectivity Factor (Vif). Vif itself is a rather small 23- kilodalton protein that plays a pivotal role in HIV replication. Vif’s main function is to counteract the antiviral activity of APOBEC3G by tagging it via an E3 ubiquitin ligase and targeting it for proteasomal degradation (Wang 2013). APOBEC3G belongs to a superfamily of APOBEC proteins, which are known to play a large role in innate anti-viral immunity (Harris 2003). APOBEC3G is a certain protein of this family that is responsible for causing hypermutations in the progeny viral DNA. It is a single stranded deoxycytidine deaminase, that, when Vif is not present, incorporates itself into the budding virion through binding of the (+) viral RNA strand. APOBEC3G functions by catalyzing the deamination of cytosine to uracil, thus a deaminase enzyme. Subsequently, this causes reverse transcriptase to transcribe the newly converted uracil as adenine in the viral RNA transcript. This mutation is sufficiently able to mutate the pro viral DNA, and inactivate it (Romani 2009).

Interaction of APOBEC3G/Vif

            The paper focuses on the processivity of APOBEC3G, and how it is affected when in the presence of Vif. Vif was primarily seen to inhibit APOBEC3G by targeting it through proteasomal degradation, as well as down-regulating APOBEC3G mRNA translation, so the ability for it to inhibit its function by another mechanism provides another pathway for anti-retroviral therapy. Past research has shown that APOBEC3G scans its substrate by both sliding and microscopic jumping around the ssDNA, providing a thorough 3D scan that increases its efficiency in locating its deamination target (Chelico 2006). In this paper, the researchers investigate the novel molecular mechanism of degradation-independent Vif mediated inhibition of APOBEC3G. Two Vif variants were used; Vif_IIIB and Vif_HXB2, both are seen to inhibit APOBEC3G deaminase activity, and in this experiment both provide a novel way in which Vif inhibits deaminase activity by successfully altering the scanning mechanism employed by APOBEC3G.

            In the first experiment, the researchers constructed a model HIV-1 replication system to measure any change in APOBEC3G mutagenesis in the presence of the Vif variants. First they performed an assay with only APOBEC3G and saw that in comparison with just reverse transcriptase, APOBEC3G caused a 17-fold increase in population mutation frequency, and a 11-fold increase in the clone mutation frequency. Having a baseline of the amount of mutations that APOBEC3G induces, the researchers then performed two additional assays, this time one of each Vif variant was introduced alongside APOBEC3G. From here they plotted the percentage of clones having a mutation in comparison with their location on the transcript. The histogram presented really makes it easy to understand the data. The histogram, Figure 1, shows that without Vif, APOBEC3G usually has a high range of induced mutations, 30% of recovered clones had 12 mutations or greater per transcript, as well as about 25% of clones showing 6-8 mutations per transcript. In the presence of Vif_HXB2, 55% of clones showed 3-5 mutations per transcript, while there was no more clones with 12 mutations or greater. The Vif_IIIB variant also showed reduction of clone mutations, to a lesser effect, by a shift down to 3-8 mutations per transcript. Together the data shows that both Vif variants are able to inhibit APOBEC3G induced mutagenesis, and their potential for being targeted for therapy.

Figure 1. Population Distribution of Mutations

Myeloid Dendritic Cells Enhance HIV Latency in T Cells

Diagram of the HIV virus

The Human Immunodeficiency Virus commonly known as HIV in the last 30 years has become a worldwide epidemic affecting approximately 23 million people with an additional 4 million cases annually.  This virus systematically disrupts and destroys the host immune system and is the direct precursor to Acquired Immunodeficiency Syndrome or AIDS.  Despite years of intense scientific research there is no cure or vaccine for HIV.  The current form of treatment is continual use of anti-retroviral drugs which can help to keep HIV at bay but are expensive and have side effects.  The virus itself invades a number of different immune cells and inserts its DNA into the DNA of the cell causing it to produce more of the HIV virus.  The virus itself is also toxic to subsets of these cells known as CD4+ T cells which die after the virus has replicated within the cell.  The destruction of these CD4+ T cells mediates the inability of the immune system to fight off other pathogens. 

                A major problem in combating HIV is that after the virus infects a cell it sometimes lays dormant in the cells to be replicated later.  In this case the virus is not immediately killing the cell but it in the meantime escapes normal processes of destruction.  This viral latency allows the virus to remain in the immune system undetected and escape destruction by other immune cells or anti-retroviral therapy.  Despite this common occurrence, the mechanisms underlying this process are relatively unknown.  However, a recent paper has partially uncovered how some CD4+ T cells are latently infected with the HIV virus.  A paper published this month by Evans et al. has demonstrated the myeloid dendritic cells are responsible for the latent infection of inactivated CD4+ T cells.  In this study, researchers co-cultured inactive CD4+ T cells with different types of infected dendritic cells to see whether they would cause a latent infection and if so which dendritic cells specifically.  The resting CD4+ cells were given a green fluorescent protein which gave off a green light when the cells were actively infected with the HIV-like virus.  The researchers then separated out the non-glowing cells which were not actively infected and further examined those.  When these CD4+ T cells were stimulated a small percentage of them turned green when they had not before, indicating that they contained the virus previously but it was not active and thus the virus was in a latent state.  This process allowed scientists to culture the CD4+ T cells with other cells or chemicals to see what factors caused the cells to have an active or latent infection.  Given this, it was soon discovered that infected myeloid dendritic cells caused a latent infection but not other types of dendritic cells.  Myeloid DCs are cells which migrate in the body and encounter pathogens.  These pathogens are then engulfed, chopped up by enzymes within the cell, and represented on the surface of the cell so that T cells may encounter the antigen and form an immune response against it.   Intriguingly it was previously thought that chemicals released by DCs or the environment called cytokines might be playing a role in the process, however, when the chemicals from the DC-T cell interaction were removed and cultured with new CD4+ T cells they did not develop a latent infection indicating that the myeloid DCs must have a physical connection to the T cells for this process to occur.  The connection between the two is an immunological synapse which involves numerous molecular adhesion molecules such as LFA-1 or ICAM.  The same study found that when they blocked these adhesion proteins there was a reduced effect of infection latency among CD4+ T cells but it did not eliminate latent infection completely so more than these two adhesion molecules must be involved.  These would be new potential targets for anti-retrovirals or other drugs.

Depiction of HIV spread from a DC to a T cell

Implications of Chronic Alcoholism for HIV Infection

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Alcohol dependence is the most common form of drug abuse in the US, with around 7% of the population meeting criteria for alcoholism (Grant 1994). We all know that the effects of alcohol are wide-ranging, impacting both behavior and physiology; it impairs judgement, motor skill, etc. It has been known to weaken the immune system, and research in the past few years has linked the effects of alcohol to HIV infection. Common sense tells us that alcohol promotes risky behaviors, including those that increase the possibility of HIV infection (sex, drugs, etc.). Recent interest in binge drinking and HIV has produced data showing alcohol in a binge pattern changed the proportion of immune cells after SIV infection in rhesus macaques, a common animal model for HIV, and may even increase the disease course (Molina et al 2006, Poonia et al 2006). But researchers at the Scripps Research Institute have also showed that chronic alcoholism may generate microenvironments in the body that are more vulnerable to HIV infection.

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To accomplish this study, the researchers developed a protocol in which rhesus macaques self-administered alcohol orally, twice a day for 30 days before infection and during infection. To do this, they mixed alcohol and an orange flavored drink in different ratios, and conditioned the animals to drink the solution (replacing water with the alcohol solution for a short duration). The final concentration given to the animals was 6% alcohol, similar to the alcohol content of most beers, and animals drank enough alcohol per day to cause blood alcohol levels that in humans are greater than the legal driving limit and would decrease motor skill. When the animals were conditioned, they were infected with SIVmac251, a strain of SIV that induces high peak and steady viral loads and is also known to infect the CNS (Burdo et al 2005). SIVs, simian immunodeficiency viruses, are retroviruses that infect non-human primates and produces symptoms and changes in physiology that mirror those induced by HIV infection (it is generally believed that HIV originated from SIV crossing the species barrier to humans).

A New Mechanism of Cell Death by HIV

            It’s a disease that is well known all over the world: human immunodeficiency virus, better known as HIV.  It is often talked about in tandem with acquired immunodeficiency syndrome, or AIDS, which develops in HIV patients over time and is the end stage of the disease.  HIV originated in chimpanzees as simian immunodeficiency virus (SIV) and transferred over into humans in the 1800s.  The first cases in the United States were reported in 1981.  Throughout the 1980s, cases increased dramatically, peaking in the early 1990s.  However, a breakthrough in drug treatment for people living with HIV and HIV prevention campaigns helped to bring the number of cases back down.  The drug therapy known as antiretroviral therapy (ART) is still used today as the main way to help people with HIV live normal lives, hopefully preventing/delaying the progression to AIDS.  They target different points in the HIV virus replication cycle to try and slow down its progression through the body.

Figure 1: HIV replication in a cell.  It is able to dump its
contents into the cell, reverse transcribe its RNA,
integrate it into host DNA, and use the host to create new viral copies.

HIV is a retrovirus, a form of RNA virus that can be inserted into the host DNA, and then uses host cells to replicate.  The virus comes with all sorts of proteins that let it do this.  For example, reverse transcriptase allows the viral RNA to be turned into viral DNA.  Integrase allows it to be inserted into the host DNA to then use the host’s own protein making mechanisms to make new viruses and viral proteins.  Figure 1 shows an outline of HIV replication.  It is possible to trace the viral RNA to viral DNA to host DNA and then back out to spread to other cells. 

HIV also specifically infects a certain class of immune cells known as CD4+ T cells.  CD4+ T cells are called this due to the presence of a cell surface receptor called CD4.  These cells can differentiate into all different subtypes of CD4+ T cells called T helper cells, or Th cells, whose name describes their function: they “help” other immune cells mount responses to pathogens.  CD4+ T cells and all of their progeny are crucial for providing immunity to all sorts of infections, pathogens, and the like.  HIV comes into play and infects the CD4+ T cells.  It creates a chain reaction, infecting, spreading, and slowly killing all the CD4+ T cells in the body.  In simple terms: HIV is slowly knocking out an entire branch of the immune system.  Any further immune function that would need a CD4+ cell to work won’t be able to work once the CD4+ T cells are gone.  Figure 2 lays this out in a graph showing CD4+ T cells in blue, viral RNA copies in read, and time in weeks on the x-axis.  It is possible to see that as the RNA copies go up, the CD4+ T cells go way down.  Many of the ART drugs target these proteins that prevent the HIV from infecting cells as easily or spreading once it has infected a cell.  However, it is still unknown how HIV actually kills CD4+ T cells.

Figure 2: Timeline of HIV infection.  As viral RNA increases,
CD4+ cells decrease.  A latent period exists where the
person may not know they are infected
until their cell count reaches a certain point.

Cooper et al demonstrate one potential way HIV could kill CD4+ T cells.  They first infected cells with HIV and stained for a particular viral protein called p24.  They noticed that the CD4+ T cells that were killed didn’t express this viral protein, while cells that weren’t killed did.  Next, they looked at whether these cells that were lacking expression of this viral protein had been infected with the virus before they died.  More T cells were infected with HIV that also encoded for green fluorescent protein (GFP), which fluoresces green.  GFP is often used as an indicator for protein production. The gene for GFP is placed within the HIV genome, so if HIV proteins are being produced, GFP will also be produced.  If the cells are dead, no GFP will be detectable.  This is a commonly used method to visualize and also quantify protein production.  They analyzed the cells for GFP expression and cell viability, as well as viral cDNA.  Non-viable GFP- cells were found to have copies of viral cDNA.  When viable GFP+ cells were watched over time, the researchers saw that many of these cells eventually died (therefore losing their GFP expression) but retained viral cDNA.  These data together suggest that the cells that were killed died after successful HIV gene expression.

Immunosupression in HIV/AIDS Mediated by Myeloid Derived Supressor Cells

MDSCs: Gabrilovich & Nagaraj, Nat Rev Immunol 2009

Recently, researchers have found a new subset of immune cells that functions to suppress immunity in multiple diseases. Early research has identified them as myeloid-derived suppressor cells (MDSCs), a heterogenous group of immature myeloid cells, divided into granulocytic or monocytic subsets (CD15 or CD14). Recent research has focused on their role in tumor immunology; Hosoi et al showed that during adoptive CTL (cytotoxic T lymphocyte) treatment of cancer (melanoma), CTLs triggered expansion of MDSCs that eventually outnumbered CTLs, secreted reactive oxygen and nitrogen species, and prevented further proliferation of CTLs. This research in cancer biology shows that immunotherapy and immune response can trigger a counter-regulatory response that dampens the immune system. In a recent paper, Ankita Garg and Stephen Spector tested the roles of MDSCs in viral infection, specifically HIV/AIDS, and found a very similar suppressive effect. As not much is known about the mechanisms behind how MDSCs function in the immune response, these authors used HIV infection of PBMCs and co-culture techniques to explore how MDSCs suppress immunity, covering their expansion, function, and mechanisms of function.

Origin of MDSCs; Gabrilovich & Nagaraj, Nat Rev Immunol 2009

To start, the authors treated PBMCs (peripheral blood mononuclear cells) with heat-inactivated or infectious HIV, or gp120, a HIV envelope glycoprotein responsible for binding to the CD4 co-receptor on a helper T cell and allowing the HIV envelope glycoprotein gp41 to contact the host cell membrane and promote viral fusion (see HIV and Neurocognitive Dysfunction). Both infectious HIV and inactivated HIV/gp120 were able to induce expansion of MDSCs (in this paper gated as CD11b⁺CD33⁺CD14⁺HLA-DR^-/lo; see note), suggesting that viral replication is not necessary of induction of MDSCs. Next, they looked that how gp120 was able to induced MDSC expansion, and found that PBMCs cultured with gp120 conditioned media showed increased an MDSC population, and found increased levels of IL-6, a major inflammatory cytokine, in gp120-treated PBMC supernatants, leading them to hypothesize that IL-6 was responsible for inducing MDSCs. They confirmed this by showing IL-6 neutralization inhibited MDSC expansion. Furthermore, they surmised that IL-6 would be acting through its STAT3 pathway (a major pathway for IL-6 signaling), and found increased levels of phosphorylated STAT3 (pSTAT3) in gp120-treated PBMCs. They showed that IL-6 neutralization completely abolished pSTAT3 expression in gp120-treated PBMCs, showing that IL-6 may cause MDSC expansion via signaling through the STAT3 pathway.

Plasmacytoid dendritic cells: Friend or Foe?

Plasmacytoid dendritic cells (look a lot like plasma B cells but express different proteins on their cell surface) are important interferon-producers in the presence of viral RNA or DNA (an interferon is a cytokine, or a signaling protein released in response to a pathogens).  pDCs are important in linking the innate (nonspecific) and adaptive (specific) immune response through increasing the function of natural killer and T cells, respectfully.

Much is still unknown about pDC function, though.  As discussed in Li et al., there is a debate on whether pDCs inhibit HIV/SIV replication or instead promote chronic immune activations and disease.  In other words, what is the role of pDCs in lentiviral (long incubation period) infections?  Using SIV (Simian immunodeficiency viruses)-infected rhesus monkeys, Li et al. tried to determine the systematic distribution of pDCs using a cell counting/sorting mechanism called flow cytometry.  They also looked their cytokine expression and affect on T cell activation upon infection. 

HIV and Neurocognitive Dysfunction

            HIV, human immunodeficiency virus, infection is one of the largest public health problems we face today. The WHO estimates that currently over 35 million people worldwide are infected with HIV. HIV primarily infects and kills helper T cells, cells that have the critical role of activating and supporting other cells of the immune system, thus impairing the immune response on a broad level. It is well known for causing AIDS, the acquired immunodeficiency syndrome, which severely weakens the body’s immune response and leaves it open to attack by foreign antigens. However, less widely known, HIV also causes neurocognitive impairment and encephalitis (swelling of the brain), estimated by Heaton et al to affect one third of individuals infected with HIV. This facet of HIV infection, which affects a whopping 10 million people worldwide, is an important concern and is now receiving more attention.

            Recently, the Section of Infectious Diseases of the Nervous System at NIH, led by Dr. Avindra Nath, and their colleagues at Johns Hopkins School of Medicine have discovered possible mechanisms for HIV-mediated neurocognitive impairment. In a recent paper in the Journal of Neurovirology, these researchers looked at the impairment of adult neurogenesis and neurite outgrowth in the hippocampus using a HIV mouse model. They believe that the growth patterns of neurons in this part of the brain, which deals with processes such as memory consolidation, may be disrupted during HIV infection and underlie the cognitive dysfunction seen in HIV infected patients. While previous research had found that HIV infects glial cells and not neurons, it was shown that HIV infection lead to synaptic pruning and apoptosis (Ellis et al 2007).

            The investigators used a transgenic mouse model that expressed the HIV envelope glycoprotein gp120 under the control of the GFAP promoter (glial fibrillary acidic protein, a protein expressed by glial cells in the brain). HIV gp120 is part of the HIV viral envelope “spike,” a device used to bind host cell receptors and enter the cell. Briefly, the spike consists of three gp120 units bound to three gp41 units; gp120 binds to the CD4 co-receptor on a helper T cell, allowing the HIV envelope glycoprotein gp41 to contact the host cell membrane and promote viral fusion. Thus, the investigators used this mouse model to simulate HIV infection, during which gp120 would be circulating in the body.

Fighting HIV-1, Marked for Death!

Based on a recent article published in January of this year in theProceedings of the Natural Academy of Science (http://www.pnas.org/content/110/11/4351.full.pdf), there may be advances in how we can mark target sites on HIV-1 virions for destruction. A main way that the body fights infection is by marking a cell for death, and then its recognition by the immune system that follows up with an antiviral response. This function of our bodies works well against most infections that we acquire through life, however HIV-1 is a true nuisance, why? Well HIV-1 belongs to a class of viruses called retroviruses. Retroviruses are different than many other viruses due to their genome being encoded with RNA. Reverse Transcriptase is essential for these viruses to produce DNA from their RNA genome, which then can be further translated to proteins. In the process of creating the DNA strand from the RNA template there is a lot of error that goes on. Because of this frequent coding error, there are usually many mutant types of protein that can either lead to increased virulence or cause no functional change at all¹. 

          In the case of cell targeting, there is usually a defined cell region that a known protein can bind to, its binding site. Once these sites are confirmed, steps can be taken to attach immunogen proteins to them, mimicking the same structure that would allow binding to the binding site. This works, however, when the defined binding region is known and readily accessible by the protein. The problem with HIV-1 is that due to its high error-prone reverse transcriptase, the coding for this binding site or the region of the virus envelope frequently changes^2,3. 

     This leads to the problem of accurately marking these sites and triggering them for destruction. Thus the researchers of this article have discovered an antibody protein that shows stable binding to the viral envelope, which can lead to engineering a vaccine, preventing viral infection.

SO WHATS THE PROTEIN AND WHAT MAKES IT SO GOOD AS A MARKER?

            Well the protein discussed is actually an antibody, and it is apart of a large family of proteins called broadly neutralizing antibodies, or bnABs. Recently these antibodies have been shown to bind to the nuclear envelope of the HIV-1 virus and effectively neutralizing it. Binding the envelope is very important since the glycoprotein found on the envelope is the mechanism by which the virus particle gains entry to the target cell. If the glycoprotein can be changed or constricted in any way (i.e. binding to it), then the virus particle is hampered from entering the cell and causing infection¹. 

        The problem is that these antibodies have mediocre binding potential due to the difficulty of binding to the conserved region on the viral envelope; with other proteins blocking the site as well as steric hindrance. However PG9, a unique bnAB, showed a stable and repetitive binding site conserved on the viral envelope. PG9 bound to the envelope in a way that was most curious, it bound with the trimers of the glycoprotein of the virus and formed a quaternary structure with the binding site. This type of binding leads to high stability, which was confirmed by the heat points recorded during the PG9-Envelope glycoprotein interaction¹. Using different viral viewing methods, such as electron microscopy and crystal structures, the researchers were able to identify the sites that PG9 binds to. The research showed that there was a high affinity in binding between PG9 and the HIV-1 envelope glycoprotein. The PG9 antibody binds to two out of the three monomers of the envelope glycoprotein, a trimer complex. This dual binding is important but other antibodies have this characteristic as well, so what is it about PG9 that makes it better? The researchers found that PG9 has a second interaction site beside the three monomers of the envelope glycoprotein, a monomer on an adjacent glycoprotein. This three monomer binding complex that PG9 forms with the envelope glycoprotein has shown to be thermally stable and readily able to neutralize virus due to its interactions at the monomers of the glycoprotein⁴. By blocking these monomers, the overall integrity of the trimers in the glycoprotein is compromised, possibly leading to its inability to enter the cell.

NOW WHAT?!

Why haven’t we found a cure for HIV?

     In 1983 the human immunodeficiency virus (HIV) was first isolated and suggested as the root cause of acquired immune deficiency syndrome (AIDS), a universally fatal condition thanks to various opportunistic diseases that take advantage of the sufferers weakened immune system (1). Today HIV is one of the most intensely researched viruses in the world and new drugs are constantly being developed to minimize its effects in HIV+ patients. Yet despite sophisticated cocktails of these drugs that are administered during the most popular treatment for HIV infection, highly active antiretroviral therapy (HAART), we still have not managed to develop a therapeutic strategy to fully eradicate the virus from those infected with it.

     The reason a cure for HIV has been so difficult to obtain has to do with the virus’s latent reservoir. Most cells that become infected with HIV start producing infectious viruses within a few days. And they do this at a high enough rate that the cell eventually dies, either directly due to the viral replication itself or indirectly due to the host’s immune system. These cells die off and are no longer a threat for producing more viruses. A cell involved in the HIV latent reservoir, however, only produces viruses at a low rate or not at all. It can remain dormant, evading the host’s immune system while still containing the HIV genome and, therefore, the ability to produce infective HIV viruses. Recent research has suggested that memory T cells, which are involved in the mechanism that allows the immune system to remember pathogens after infection has cleared, make up the largest proportion of the HIV latent reservoir (P,2). The long-lived nature of this cell type means that it would take an exceedingly long time to wait for each of these proviral cells to die. Recent modeling suggests it could take up to 70 years (3).

     Because so many successful antiretrovirals have been developed, patients adhering to HAART can delay the onset of AIDS indefinitely (P). These drugs are very good at preventing any HIV viruses that persist in the patients bloodstream from infecting new CD4+ T-cells, the viruses target cell type. However, once taken off HAART, these patients start shedding new viruses and progress rapidly to AIDS. Therefore, HAART must be a lifelong treatment, one that is very expensive and very difficult to keep up with. The reason HAART does not fully eradicate HIV is because the cells of the latent reservoir still contain the HIV genome, which enables those cells to manufacture infectious viruses, which are detected in the individuals blood. If the patient is fully adhering to HAART these viruses simply degrade and do not infect new cells. But if the patient is taken off HAART these viruses can infect new cells, which can then go on to shed more viruses.

Hope for an HIV Vaccine

In 1984, the Health and Human Services Secretary declared that extensive research for a vaccine protecting against Human Immunodeficiency Virus, a virus that ravishes the immune system of its host, would be available in two years. Almost three decades and one billion dollars later, scientists have still not been able to develop a vaccine that can successfully protect against the virus. The reasoning for these difficulties revolve around HIV's ability to rapidly mutate, making it near impossible for the immune system to mount an effective response (class notes, 12/8/2011).

During a normal immune response, an immune system cell called a B-cell makes antibodies, a protein that neutralizes specific proteins on a pathogen called antigens, preventing it from infecting the host. When a person gets vaccinated against a pathogen, their immune system is being given a form of the pathogen that does not have the ability to infect the host. This form of the pathogen allows the host's immune system to prime itself against the virus, which includes the creation of antibodies, so that when the host comes in contact with the infectious form of the pathogen, the antibodies will be waiting ready to stop the it.

However, the problem with HIV is not that it stops the body from making these antigens, but that it mutates so quickly that it is able to circumvent the antibodies. When an antibody is made, it will bind to a specific part of the outside of the pathogen, but once antibodies against HIV are made, the virus has already changed its external characteristics such that the antibody cannot bind to the virus and neutralize it. This is the main reason why making a vaccine against HIV has been so difficult.

Instead of injecting mice with parts of the virus in hopes of causing the creation of antibodies against HIV, Alejandro Balazs used a technique called vectored immunoprophylaxis (VIP). This involves inserting DNA for an HIV antibody into a virus known to be harmless to humans, called adeno-associated virus (AAV), and then injecting this virus into the host's muscle, in the hopes that it will cause the host to make antibodies that will neutralize HIV. These scientists injected VIP into mice and found that the mice were fully protected against the HIV virus.

Helpful Hormones: The potential benefits of hormonal contraceptives on delaying HIV progression to AIDS

Any person who has sat through “sex-ed” has heard about practicing safe sex, using condoms to prevent the transfer of STDs from one partner to another. Additionally, there are other methods of contraceptives, namely hormonal contraceptives. Once one has contracted HIV there is an even increased necessity to protect against pregnancy in order to prevent transmission of disease to the child and to protect the mother against unwanted pregnancy. However, with hormone treatments there have been studies that indicated the hormonal contraceptives actually advance the progression of HIV in patients.¹ While this has mainly been documented in undeveloped countries, any implication in the advancement of HIV towards AIDS is a concern and should be studied.

Some of the largest populations of HIV positive persons are the commercial sex workers and the populations in sub-Saharan Africa.² One study in particular followed a group of HIV seroconverted women located in Uganda for a person of 11 years. This was one of the most extensive studies with one of the largest sample sizes, thus increasing the possibility for significant results (625 women in study). The women in this study were compared for differences between the hormonal contraceptive users and nonusers and monitored for the change from HIV to AIDS or death. Hormonal contraceptives in this study were considered both oral contraceptive pills and implanted hormonal contraceptives.

In the entire study 27% of participants used hormonal contraception at some point during the study. What the researchers found was that, in general, women who reported using hormonal contraceptives had a longer delay to onset of AIDS and a longer time until death. Specifically, those who used hormonal contraception had on average 3.92 years until death, and 3.72 years until AIDS, compared to 3.04 years until death and 2.98 years until AIDS or those without hormonal contraceptive use. Interestingly, this study also took note as to what kind of behavioral patterns the hormonal contraceptive users engaged in compared to the nonusers. This study found that women using hormonal contraceptives had on average a higher degree of education, were in the median age category (25-35), and were also less likely to use condoms. These have to be taken into account as well as the hormonal contraceptive use as possible variables affecting onset of AIDS and death.

While there have been studies that have linked hormonal contraceptives with an increase in time to onset of AIDS, the results of this study are not unheard of. Some previous studies have suggested that the hormonal contraceptives may actually reduce HIV progression.³ This study was also quick to point out their strengths, namely that their study provides evidence that hormonal contraceptives do not have an impact on the progression of HIV to AIDS/death. However, there were also limitations that this study had to deal with and should be pointed out in this discussion. In particular, it was very difficult to determine a death as AIDS related as opposed to outside causes. Another problem with this study was the difficulty in determining the varying affects that differing hormonal contraceptives had on varying the degrees of HIV progression.

The big picture to take away from this study is the vast array of treatments and possible drug therapies that can be applied to HIV/AIDS patients. There are large groups of people worldwide who are HIV seroconverters that are being observed and studied in order to advance preventive technologies. It is important that all possible treatments are considered as there has yet to be an effective vaccine developed. While the specific biological interactions were not considered in this study, there is still a future direction in hormone treatment, and anything that can slow the onset of AIDS is worth a second look.

Primary Resource

Polis C, Wawer M, Kiwanuka N, Laeyendecker O, Kagaayi J, Lutalo T, Nalugoda F, Kigozi G, Serwadda D, and Gray R. (2010). Effect of hormonal contraceptive use on HIV progression in female HIV seroconverters in Rakai, Uganda. AIDS. 24:1937-1944. Doi: 10.1097/QAD.0b013e32833b3282

Additional Resources

1. Stringer EM, Kaseba C Levy J, Sinkala M, Goldenber RL, Chi BH, et al. (2007) A randomized trial of the intrauterine contraceptive device vs hormonal contraception in women who are infected with the human immunodeficiency virus. Am J Obstet Gynecol 197: 144-148

2. “UNAIDS: Sub-Saharan Africa” WHO. 2009 091124_fs_ssa_en.pdf>

3. Allen S, Stephenson R, Weiss H, Karita E, Priddy R, Fuller L, et a. (2007) Pregnancy, hormonal contraceptive use, and HIV-related death in Rwanda. J Womens Health. 15:1017-1027

CD4+ T cells in HIV

Human immunodeficiency virus (HIV) is a member of the retrovirus family and progresses into AIDs after a period of time. AIDs is defined by a significant deficiency in T-cell count which causes a progressive failure of the immune system and allows life-threatening opportunistic infections and cancers to thrive. Infection with HIV occurs by the transfer of blood, semen, vaginal fluid, or breast milk. In order to slow the progression to AIDs, many people with HIV take antiretroviral therapy which combines a number of drugs that are designed to stop HIV from infecting cells: includes nonnucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors, and triple-nucleoside (or nucleotide) reverse transcriptase inhibitors (NRTIs) (1). HIV initially depletes the body’s CD4 T-cells and the point of this therapy is to boost the immune system back to normal to continue to fight the infection. However, 30% of patients with HIV who receive antiretroviral therapy fail to achieve a normal CD4⁺ T-cell count (2). This failure to achieve normal T-cell levels results in a steady decrease in T-cells to where, in less than 10 years, the count is below 500 (signifying AIDs) leaving the patient open to opportunistic infections and eventually death. Therefore it is necessary to study the reasons for this initial failure to reach normal T-cell counts with antiretroviral therapy in order to enhance its function.

HIV first infects CD4 T-cells, depleting the amount in the body. However, there is normally a period of time in which the T-cells regenerate in order to fight the infection. The balance of T-cells in the body is negatively regulated by interferon α (IFN-α) and evidence suggests that HIV increase the production of this interferon (3). This regulating interferon may promote apoptosis of uninfected CD4⁺ T cells by up-regulating expression of a death signal (TRAIL) and its death receptor (TRAIL receptor) (4). Therefore, the role of IFN-α may have important effects on CD4 T-cell populations in HIV patients.

A study by Sonia Fernandez at the School of Pathology and Laboratory Medicine in Australia looks at the effects of IFN-α on CD4 T-cell populations in HIV patients. They recruited HIV patients who have been receiving effective doses of antiretroviral therapy and measured the levels of CD 4 T-cells in the blood as well as levels of IFN-α. The patients were divided into low or high CD4 T-cell groups. Expression of markers of T-cell activation (HLA-DR), apoptotic potential (Fas), or aging (CD57) were assessed and it was found that the proportions of CD4⁺ T cells expressing HLA-DR or Fas were higher in patients with low CD4⁺ T-cell counts than in those with high counts such that the higher the levels of Fas and HLA-DR that are expressed, the lower the population of CD4+ T-cell count. The proportion of CD4⁺ T cells expressing CD57 did not differ between patients with low or high CD4⁺ T-cell counts. They left the cells in a culture for 72 hours and found that the proportions of CD4⁺ and CD8⁺ T cells that were apoptotic or preapoptotic was similar in patients with low and high CD4⁺ T-cell counts.

HIV and Malaria Co-Infection: A Game of Immunological Russian Roulette

Around the world, there are approximately 300 million cases of malaria, from which 1 million deaths occur (1). In 2010 alone, the number of people infected with HIV increased by 2.7 million, altogether amounting to 36.7 million HIV-infected individuals globally (2). Both “diseases of poverty”, malaria and HIV have similar geographical distributions, prevalent particularly in third-world sub-Saharan Africa, the Indian subcontinent, and Southeast Asia. Studies have shown that due to this geographical overlap, co-infection of the two diseases is a common phenomenon in these regions (3,4).

HIV/AIDS is brought about by a virus whereas malaria is caused by a plasmodium. Co-infection of the two diseases, therefore, has great potential towards the investigation of dynamics between the mechanisms of immune response to both infections. Ryan-Payseur, et al., in this paper, are looking to elucidate just that; that is, what exactly happens to the immune system when it is infected by a virus and a parasite? This is an interesting paper because in research, it is difficult to examine the effects of co-infection in naturally occurring infections because naïve individuals do not exist—humans co-infected with HIV and malaria often have been previously exposed to malaria, which can complicate results (5). Nevertheless, the researchers for this paper were able to circumvent this problem, as well as the additional problem of an accurate animal model (Fig. 1), through a series of imaginative tweaks to the primate model of Simian Immunodeficiency Virus (SIV)-plasmodium infection.

[ Fig. 1 ]

HIV-1 Integration is Reduced in CD4 T Cells from Elite Controllers

In 2010, 34 million people were living in the world with Human Immunodeficiency Virus (HIV) (1). The current reality, however daunting, is that there are no vaccines available against HIV. Indeed, even modern day treatments are unable to stop progression of HIV to acquired immunodeficiency syndrome, or AIDS. In 2010, 1.8 million people died from AIDS, so clearly this is a high priority area of research (1). Scientists are working all over the globe, continually researching and studying HIV in hopes of developing a vaccine. So far the efforts have been met with continual challenges and not much success. However, there is a group of people referred to as elite controllers, who are able to maintain undetectable levels of HIV-1 replication despite being HIV positive. One could understand why they would be of great interest to scientists, as they provide a model to study for an effective immune response against HIV infection.

A recent article studying elite controllers investigated HIV-1 reverse transcription and integration in directly ex vivo­ isolated CD4 T cells (2). They used CD4 T cells from elite controllers, HIV-1-negative volunteers, and untreated HIV-1 progressors. The quantified CD4 T cell count using negative immunomagnetic selection ex vivo. Next, cells were infected with a yellow fluorescence protein (YFP)-encoding vesicular stomatitis virus G protein (VSV-g) pseudo typed HIV-1 virus. These big words may sound confusing, but just know that this process infects cells, causing only a single round of HIV-1 infection, allowing for detailed assessments of individual early HIV-1 replication steps. Understanding these processes could have very important implications for vaccine development.

Debunking the Doublet: Study Finds Lack of Suggested Relationship Between IL-7 and CD4 T Cell Populations in Longitudinal Assessment of HIV Patients

Seventy-two patients with HIV participated in a recent study done by the European Union to investigate the correlation between IL-7 and CD4 T cell population density. The results of the study will be published in the December 15, 2011 edition of the Journal of Acquired Immune Deficiency Syndromes (vol 58, issue 5, pages 436-441), but is published in advance online. Thirty of the patients did not use anti-retroviral drugs and forty-two did, making up the two populations used in the study. T cell and IL-7 counts were taken regularly for 46 +/- 14 months in the two populations. The researchers then used multi-variable linear regression analysis to analyze the statistical significance of the potential IL-7/CD4 T cell correlation.

HIV is an acronym for Human Immunodeficiency Virus, a retrovirus that infects humans and is transferred through the direct sharing of bodily fluids through sexual intercourse, blood transfusions, or the sharing of syringes. There is no known cure or vaccine for HIV, and there are several challenges that make such a necessary product difficult to formulate. Primarily, HIV is a quickly mutating virus, which ensures that the virus readily mutates to avoid any single drug that is aimed against it. Another difficulty encountered when attempting to make and test a potential drug is that there currently exists no appealing animal model in which to test HIV treatments and vaccines. Chimpanzees respond to SIV, a virus similar to HIV but that infects chimps, but they never progress to AIDS. Also, chimpanzees are an endangered species, which makes using them as an animal model ethically questionable and difficult to obtain approval for.

Cytheris, a biopharmaceutical company that works with immune enhancement, has had success in mouse trials using IL-7 to treat HIV, and is currently working on Phase I clinical trials with a new HIV drug based on these results.¹ This has led to much investigation of the link between IL-7 and HIV, one outcome of which is this study. CD4 T cells are often the first immune system element that comes to mind when pondering HIV, as these are the main targets of the virus. HIV infects and brings about the death of CD4 “helper” T cells, and when the CD4 T cell count falls below 200 cells per cubic mm of blood the patient is considered to have progressed from HIV to AIDS. Thus, CD4 T cells were suggested as the link between HIV treatment and IL-7. However, the perhaps most likely link between IL-7 and the treatment of HIV, CD4 T cells, did not produce the expected results.

Dodging Natural Killing: HIV Innate Immune Evasion

Image via Wikipedia Commons

HIV, the virus that causes AIDS, is the cause of one of the most far-reaching and destructive epidemics in modern times. Responsible for approximately 1.8 million deaths and more than 2.5 million new infections annually, HIV is a major public health concern, particularly in the developing world. HIV-1, the most common and virulent strain of the virus, has been studied extensively by researchers hoping to better understand the virus’s life cycle and develop treatments that disrupt its ability to infect and reproduce. One of the greatest barriers to effective treatment and clearance of HIV-1 is the virus’s astonishing capacity for immune evasion. From constant mutation to active subversion of the body's immune system, HIV is a particularly difficult pathogen to eliminate. In a paper published in PLoS: Pathogens in March of 2010, researchers from the Institut Pasteur examined one method by which HIV-1 is able to evade the natural killer (NK) cells of the innate immune system and establish a persistent infection, providing both a better understanding of HIV-1 immune evasion and a possible target for future HIV treatments.

Under normal conditions, viruses and virus particles are taken up by a subset of the host’s white blood cells known as dendritic cells (DCs). The DCs act as the immune system’s sentries, scouting the body’s tissues for foreign particles or pathogens and presenting them to other immune cells to stimulate a response. DC function is closely regulated by the interactions that take place with NK cells following infection. When inflammation occurs at the site of an infection or injuries, chemical signals known as chemokines cause NK cells to migrate to the infection site. Upon arrival, NK cells interact with DCs, sending signals that may cause the DCs to mature, and receive activating signals from the DCs. If an NK cell encounters a DC that has been infected by a virus or other intracellular pathogen, the NK cell will kill the infected cell by inducing a process known as apoptosis. When the infected DCs undergo apoptosis – also known as programmed cell death – the virus inside them can no longer replicate. This process is essential to the immune system’s ability to clear a virus from the body, particularly during the early stages of infection. However, previous research has suggested that HIV positive people are deficient in killing DCs that are infected with HIV-1, causing the infected DCs to act as a reservoir in which the virus can replicate without interference. In this paper, the researcher set out to determine the methods by which HIV-1 is able to prevent NK cells from killing infected DCs.