Looking closer at HIV infection and immunity
For more than 20 years, Alexandra Howell, a Geisel professor of medicine and of microbiology and immunology, and a researcher at the White River Junction VA Medical Center, has been working to understand the mechanics of HIV infection. Her particular focus has been studying the conditions in the female reproductive tract that either contribute to or inhibit infection. She notes that although the likelihood that a woman will become infected with HIV following sexual contact with an infected partner is fairly low, in the range of 1 in 400 to 1 in 1000 encounters, certain conditions can increase susceptibility. Worldwide, the number of women with HIV has grown steadily in recent years.
Howell wants to identify the conditions that promote or prevent infection in the hope of developing more effective measures to decrease the risk for women. An early step for Howell was to examine the role of the reproductive hormones estradiol and progesterone, which fluctuate during the menstrual cycle, pregnancy, and menopause. By studying blood cells in vitro, she found that high levels of estradiol protected cells against infection, whereas high levels of progesterone seemed to promote it. Then, about four years ago, her research team took their investigation to the next level by studying how to block infection regardless of hormonal conditions. "We wanted to block expression of the proteins that HIV needs to get into a cell," Howell says. To infect a cell, HIV has to bind to two proteins: CD4 and CCR5. She hoped that by blocking the expression of those proteins, the virus would be prevented from entering the cell and the immune system would be able to destroy the virus.
The researchers attempted to block production of CD4 and CCR5 in mice with a humanized immune system using a method called RNA interference. This technique involves using small pieces of short interfering RNA (siRNA) that bind to the messenger RNA (mRNA) that codes for CD4 and CCR5, blocking production of the proteins.
Howell and her team put the siRNA that blocks CD4 and CCR5 into the vaginal tracts of the mice, waited three days, then infected the mice with HIV and monitored the bloodstream for evidence of infection. A control group of mice received siRNA that was "irrelevant"—it was the same size as the CD4 and CCR5 siRNA, but it didn't bind to any mRNA in the cells.
We were scratching our heads, wondering what was going on.
As they had hoped, the researchers found that the siRNA that blocked expression of CD4 and CCR5 inhibited the transmission of HIV very effectively. But what surprised them, she says, was that the control group—mice that received the irrelevant siRNA—were also protected from HIV infection. "We were scratching our heads, wondering what was going on," Howell says. The receptors should not have been suppressed, yet the control mice also had very low levels of infection compared to mice that were not treated at all.
To tease out the reason the mice that received the ostensibly ineffective siRNA were protected, the researchers added siRNAs to human blood cells in the lab. They discovered that the short pieces of siRNA looked like the genome of HIV to the immune cells. "This made sense, since HIV uses RNA for its genome," says Howell. They found that the cells were taking up siRNA and responding as if it were a virus, secreting innate immune factors. "It didn't matter what the sequence of the siRNA was, it was inducing these same protective immune cytokines," she says—namely, a cytokine called interferon-alpha. Howell's group hypothesized that Toll-like receptors, or TLRs, were becoming activated in the immune cells that took up the siRNA, because they knew that some TLRs are specific for small pieces of RNA.
In a recent study published in the journal AIDS Research and Human Retroviruses, Howell and her team tested whether the specific TLR that becomes activated by RNA, TLR7, was involved by using a compound called gardiquimod, a drug known to bind to and activate TLR7. What they found was "strange," says Howell: "The cells treated with gardiquimod were almost impossible to infect with HIV." Why? They confirmed that the cells were cranking out interferon-alpha. But even when they blocked production of interferon-alpha, the cells were still resistant to infection, albeit less so. What Howell discovered is that gardiquimod also stops the action of an HIV enzyme called reverse transcriptase, which the virus needs to convert its RNA to DNA, preventing its replication machinery from being inserted into the chromosome of the cell, thus preventing infection.
Now that the researchers have made this discovery, Howell thinks it's possible that eventually gardiquimod or a similar compound might be used as a microbicide. But first she wants to find out exactly how gardiquimod works.
"The discovery part is interesting—that's the sizzle with your steak," she says. But, she adds, what's really important is understanding infection, and figuring out how to prevent it.
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