HIV


by Roseann Vorce, Ph.D., 
Department of Pharmacology and Toxicology, Michigan State University 

AIDS Journal

AIDS Journal
July 17, 2010 – Volume 24 – Issue 11

A report earlier this month describing a functional cure of HIV infection in a baby created quite a stir among scientists, health care workers, AIDS activists, and the general public. It was billed as the first time that HIV had been essentially eradicated from the body, giving hope to millions of people infected with HIV. As it turns out, a number of adults also have experienced a functional cure of HIV infection. In some ways, the functional cure of these HIV+ adults, termed post-treatment controllers, is more remarkable than is the functional cure of the infant.

As reported in the journal AIDS, 14 adults who had been infected with HIV have kept the virus under control for as long as 9.5 years after standard antiretroviral therapy was interrupted. The median time elapsed since therapy interruption was ~7.5 years, which is nearly 4x longer than the baby has remained HIV-free. In addition, the majority of these post-treatment controllers exhibited symptoms of infection prior to initiation of drug therapy, confirming that they truly had been infected with HIV. In contrast, some scientists remain skeptical that the baby truly had been infected with HIV, although several lines of evidence indicate that she harbored the virus. Researchers can find no sign of intact virus in the baby, whereas the adult post-treatment controllers retain low levels of intact virus. A few of the post-treatment controllers have experienced transient spikes in their viral loads. These temporary increases in detectable virus indicate that HIV can replicate, but the quick return to extremely low levels of virus confirm that the body can control the infection. Thus, the HIV status of these post-treatment controllers is different from that of the baby, although they share distinction of being functionally cured of HIV.

The authors of the AIDS article have explored several explanations for the ability of the post-treatment controllers to keep the virus in check. Although they have not determined a definitive answer, two major factors appear to be necessary, although not sufficient, to produce HIV control post-treatment: early treatment using a combination of antiretroviral drugs.

  1. Early antiretroviral therapy. All 14 patients started taking antiretroviral drugs during primary HIV infection; therapy was started within 10 weeks of infection. This early treatment appears to have restricted both the number and distribution of viral reservoirs in the body. In other words, combination antiretroviral therapy limited the places where HIV could hide.
  2. Combination antiretroviral therapy. Physicians had treated each of the 14 patients with a combination of antiretroviral drugs, including nucleoside reverse transcriptase inhibitors (e.g., zidovudine and lamivudine), non-nucleoside reverse transcriptase inhibitors (e.g., nevirapine), and protease inhibitors. Most patients received drugs from two or more classes, but two patients received only two or three different nucleotide reverse transcriptase inhibitors.

As was the case of the baby cured of HIV, physicians had treated these post-treatment controllers using standard combination antiretroviral therapy; no new drugs or novel treatment protocols were used. Because this study was retrospective in nature, the treatment regimens of the post-treatment controllers differed. These differences make it almost impossible to identify relevant factors that contributed to the functional cure. In the future, carefully designed studies might enable researchers to develop a drug treatment protocol that maximizes the probability that patients will become post-treatment controllers. Hopefully, the right combination of drugs, administered during primary infection, at the right dose, and for the right duration, will increase the proportion of HIV+ patients who can control the virus without a lifetime of antiretroviral drugs.

  • Sáez-Cirión A, Bacchus C, Hocqueloux L, Avettand-Fenoel V, Girault I, Lecuroux C, Potard V, Versmisse P, Melard A, Prazuck T, Descours B, Guergnon J, Viard J-P, Boufassa F, Lambotte O, Goujard C, Meyer L, Costagliola D, Venet A, Pancino G, Autran B, Rouzioux C and the ANRS VISCONTI Study Group. Post-treatment HIV-1 controllers with a long-term virological remission after the interruption of early initiated antiretroviral therapy ANRS VISCONTI Study. PLOS Pathogens. 9, e1003211, 2013.
  • Hocqueloux L, Prazuck T, Avettand-Fenoel V, Lafeuillade A, Cardon B, Viard J-P, and Rouzioux C. Long-term immunovirologic control following antiretroviral therapy interruption in patients treated at the time of primary HIV-1 infection. AIDS. 24:1598–1601, 2010.
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http://en.wikipedia.org/wiki/Melittin

Artist and scientist Julian Voss-Andreae has created a sculpture based on the structure of melittin, which he mentions in a talk about his work. (http://en.wikipedia.org/wiki/Melittin)

by Roseann Vorce, Ph.D., 
Department of Pharmacology and Toxicology, Michigan State University 

In addition to last week’s news about a functional cure of HIV in an infant, a paper describing the ability of bee venom to destroy HIV was published in the journal “Antiviral Therapy”.  Anyone who has been stung by a bee is probably cringing at the prospect of receiving bee venom, because bee stings hurt!  Much of the pain you experience from a bee sting is caused by melittin, the primary active component in bee venom.  HIV researchers have taken the sting out of melittin therapy by cleverly packaging this toxin in a form that prevents it from reaching your cells.  This breakthrough finding represents a change in the form of melittin, not in its function.  By manipulating the form of melittin, it has been converted from a general toxin to a virus-specific drug.  The concept of controlling the function of a compound by changing its physical form represents a novel way to develop new drugs from old compounds.

Before we get into the bioengineering aspects of this development in HIV prevention, let’s first discuss the mechanism by which melittin destroys HIV.  Melittin is a small protein consisting of 26 amino acids.  The hydrophilic carboxy-terminus accounts for its water soluble nature, whereas the hydrophobic amino terminus promotes its spontaneous and rapid interactions with phospholipid membranes.  These interactions damage membranes in a nonspecific manner, severely compromising membrane structure and function.  Some viruses, including HIV, are surrounded by a lipid membrane called the viral envelope, and such viruses require an intact viral envelope to infect cells.  If the lipid envelope of HIV is disrupted, HIV is rendered non-infective.  By virtue of its ability to damage membranes, melittin disrupts the viral envelope surrounding HIV, thereby destroying the ability of HIV to infect cells.  For this reason, scientists have studied melittin as a chemoprophylactic substance that can inhibit infection of HIV and disrupt the transmission of this virus.

Although melittin destroys the infectivity of HIV particles, the utility of this toxin is limited by its nonspecific cytotoxic effects:  melittin kills cells by disrupting membrane structure and function.  If administered directly to humans, melittin would kill any cell it encounters, causing widespread tissue damage.  Therefore, the researchers needed to develop a method to deliver melittin so that it comes into contact with HIV particles, but not human cells.  Although this problem appears to be nearly insurmountable, the recent developments in nanoparticle-mediated drug delivery provided the investigators with a tool to limit contact with melittin that is based on the large size difference between tiny viral particles and comparatively large human cells.

Nanoparticles are extremely small particles that can be engineered to exhibit precise physicochemical properties.  In recent years, scientists have harnessed nanoparticles to deliver drugs and other bioactive substances to solve challenging pharmacokinetic problems.  Nanoparticles used for drug delivery, which typically range in size from approximately 1 to 100 nanometers, can be composed of a variety of pharmacologically inert materials.  Drugs attached to the nanoparticles can gain access to any compartment available to the nanoparticle; through careful engineering, nanoparticles can be targeted to specific cell types, tissues, or organs.

https://i1.wp.com/news.wustl.edu/news/PublishingImages/HIVnano_primary.jpg

JOSHUA L. HOOD, MD, PhD – Nanoparticles (purple) carrying melittin (green) fuse with HIV (small circles with spiked outer ring), destroying the virus’s protective envelope. Molecular bumpers (small red ovals) prevent the nanoparticles from harming the body’s normal cells, which are much larger in size. (click on image to be directed to Washington University in St. Louis article.)

In the Antiviral Therapy paper, HIV researchers used nanoparticles to destroy the infectivity of HIV by allowing viral particles access to melittin, while excluding human cells.  First, they created nanoparticles composed of a perfluorocarbon (PFC) core and “molecular bumpers” made of polyethylene glycol (PEG).  Next, they attached melittin to the PFC core.  In the presence of human cells, the large PEG molecules on the outside of the nanoparticle physically prevent the large cells from reaching the core surface, effectively preventing interactions between the cell membranes and the melittin located on the nanoparticle core.   Because HIV viral particles are much smaller than the human cells, the virus readily slips between the bumpers and comes into contact with melittin.  Upon contact, the melittin disrupts the viral envelope, and the HIV loses the ability to infect the cells.  Using this strategy, researchers demonstrated that these melittin nanoparticles dramatically decrease HIV infectivity, without harming human cells. In other words, through careful engineering of melittin nanoparticles, scientists have blocked human cells from contact with melittin, while allowing this toxin to interact with and inactivate HIV.

Melittin nanoparticles represent an advance in HIV therapy because this approach carries the potential for future development as a prophylactic agent.  In contrast, traditional antiretroviral agents inhibit viral replication within human cells to decrease viral load, but they cannot stop HIV from infecting human cells.  By destroying HIV infectivity, melittin nanoparticles can prevent HIV infection.  Researchers envision development of a vaginal gel containing melittin nanoparticles that will prevent the transmission of HIV between partners who wish to become pregnant or in areas where cultural norms prevent condom use.  Of course, a vaginal gel does not provide protection for people engaging in alternative sexual practices or who risk contracting HIV through intravenous drug use.  However, because melittin is an agent that destroys an essential component of the HIV virus, this toxin also holds the potential to cure HIV.  Researchers have speculated that melittin nanoparticles might someday be administered as an intravenous injection, able to completely clear HIV from the body.  In the future, using a combination of traditional antiretroviral drugs, novel therapeutic agents and treatment regimens, and new approaches for drug delivery, perhaps HIV will become a curable disease.

References:
Hood JL, Jallouk AP, Campbell N, Ratner L, and Wickline SA.  Cytolytic nanoparticles attenuate HIV-1 infectivity.  Antiviral Therapy 18:95-103, 2013.

http://news.wustl.edu/news/Pages/25061.aspx

Faraji, AH and Wipf, P.  Nanoparticles in cellular drug delivery.  Bioorganic & Medicinal Chemistry 17:2950-2962, 2009.

by Roseann Vorce, Ph.D., 
Department of Pharmacology and Toxicology, Michigan State University 

If you’ve noticed the headlines, and they’ve been almost impossible to miss, you’ve probably wondered how HIV researchers cured an HIV-infected infant.

  • “Baby Cured of HIV for the First Time, Researchers Say” (Wall Street Journal)
  • “Baby cured of HIV offers hope” (USA Today)
  • “US doctors cure child born with HIV” (The Guardian, UK)
  • “Baby’s HIV “cure” differs from previously reported case” (CBS News)

These headlines are based on a paper presented at the 20th Conference on Retroviruses and Opportunistic Infections. According to the report, physicians effected a functional cure of an HIV-infected infant using aggressive antiretroviral drug therapy initiated shortly after birth. This news is exciting because this baby is the first person to have been cured of HIV through drug therapy. Although you might suspect that a novel anti-HIV drug was responsible for the baby’s cure, she was initially treated with three standard antiretroviral drugs: zidovudine, lamivudine, and nevirapine.

Before we take a closer look at the case of the groundbreaking cure, let’s examine the three drugs used in this patient.

Zidovudine

Zidovudine

Zidovudine, lamivudine, and nevirapine all inhibit the HIV enzyme reverse transcriptase (RT). This enzyme converts viral RNA into DNA, and inhibition of RT inhibits HIV replication. By combining drugs that use complementary mechanisms to inhibit RT activity, physicians have been able to dramatically decrease the viral load in HIV positive patients. However, this drug combination has never before eliminated HIV from an infected person.

Zidovudine (AZT) competitively inhibits RT because this drug is an analog of thymidine. When RT incorporates a zidovudine molecule into the newly synthesized DNA strand, chain elongation is terminated, and an incomplete DNA molecule is produced. Similarly, lamivudine inhibits RT because this drug is an analog of deoxycytidine. DNA chain elongation is blocked when RT incorporates a lamivudine molecule into DNA, again producing an incomplete DNA molecule. Due to its rapid mutation rate, HIV readily develops resistance to both zidovudine and lamivudine. However, the RT gene only rarely acquires mutations that confer resistance to both drugs. Therefore, simultaneous administration of these two competitive inhibitors inhibits HIV replication and delays the development of resistance, resulting in a decreased viral load in HIV positive patients.

Lamivudine

Zidovudine and lamivudine are nucleoside prodrugs that are well-absorbed after oral administration. Both drugs readily cross cell membranes to reach the intracellular compartment. Once drug molecules reach the inside of a cell, they are phosphorylated to the triphosphate form, which is the active form of the drugs. In addition to serving as the active drug species, the phosphorylated forms of zidovudine and lamivudine cannot diffuse back through the cell membranes; in essence, phosphorylation traps the drug at its site of action within cells. For this reason, zidovudine and lamivudine remain active even after plasma drug levels have fallen dramatically. Therefore, once daily dosing produces adequate pharmacological activity.

The third drug, nevirapine, also inhibits RT activity, but this non-nucleoside RT inhibitor uses a different mechanism. Instead of competing with wild type nucleotides for the catalytic binding site, nevirapine binds to a separate allosteric binding site on RT. Upon binding, nevirapine alters the conformation of the catalytic site of the enzyme, thereby inactivating it. After oral administration, nevirapine exhibits good bioavailability and readily penetrates the cell membrane. The cytochrome P450 system metabolizes this drug to produce several metabolites, which are subsequently glucuronidated and excreted in urine. Nevirapine induces its own metabolism, requiring patients to increase the dose after two weeks on the drug. HIV readily develops resistance to nevirapine, and co-administration of this drug with other RT inhibitors improves efficacy and inhibits the emergence of drug-resistant strains.

nevirapine

Nevirapine

Now that we know which drugs were used, let’s return to the baby girl who was cured of HIV.
She was born to an undiagnosed and untreated HIV positive mother. Consequently, she was treated almost immediately with an aggressive regimen of zidovudine, lamivudine, and nevirapine. A week later, the combination of lopinavir (inhibits HIV protease) and ritonavir (inhibits the P450 that metabolizes lopinavir) replaced the nevirapine. After 18 months of treatment, therapy was discontinued. Because cessation of drug therapy typically causes the retroviral load to rebound, HIV researchers were elated that the child tested negative several months later! Using ultrasensitive methods to detect HIV RNA, researchers found a vanishingly small number of RNA copies, but they detected no replication competent virus. Therefore, the initial therapy appears to have cured this child of HIV infection, and she remains HIV negative.

If the drugs used to treat the infant weren’t new, then what was the difference responsible for the functional cure? The simple answer is that the physicians initiated therapy using an aggressive treatment protocol in lieu of a more conservative prophylaxis protocol. Instead of administering one or two RT inhibitors to prevent the baby from becoming infected, doctors administered all three RT inhibitors starting almost immediately (31 hours after birth). This treatment protocol was initiated because the mother had not received prenatal care designed to minimize transmission of HIV to the infant. When the mother’s positive HIV status is known in advance, she receives anti-HIV therapy during pregnancy, which dramatically decreases the infant’s infection rate. In addition, the infant is treated with a postnatal drug regimen designed to prevent infection. In this case, the mother received no prenatal care, and her positive HIV status was not discovered until she was in labor. The physicians’ decision to treat the baby aggressively paid off: tests confirmed that she had been infected prior to birth, and the treatment regimen has produced a functional cure of her HIV infection.

Although some scientists have suggested that the infant was not truly infected, the fact remains that the baby tested HIV-positive shortly after birth, responded in a typical fashion to antiretroviral therapy, received treatment for only 18 months, and has tested negative from ages 24-26 months. This unprecedented finding suggests that a functional cure of HIV infection is within our grasp, if the right combination of antiretroviral drugs is administered at the right time and at the right doses. This report also demonstrates that scientific progress often occurs incrementally, using known tools in a different way, administering standard drugs using a different protocol, or by a physician violating standard treatment protocol when the conditions surrounding patient care are not standard.

————–
References:
Persaud D, Gay H, Ziemniak C, Chen YH, Piatak M, Chun T-W, Strain M, Richman D, and Luzuriaga K. Functional HIV cure after very early ART of an infected infant. 20th Conference on Retroviruses and Opportunistic Infections, Paper #48LB, 2013. Accessed on 06 March 2013 from:  http://www.retroconference.org/2013b/Abstracts/47897.htm

http://www.niaid.nih.gov/news/newsreleases/2013/Pages/toddlerfunctionallycured.aspx

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