Scientists Report New Clues on How HIV Enters Human Body

Scientists have discovered that people who are sexually exposed to HIV-1 receive two types of the virus, known as M-tropic and T-tropic. They also have determined that only the M-tropic variety seems to be able to establish infection after sexual contact.

Researchers say this is puzzling because the immune cells that first encounter HIV-1 on genital surfaces, called Langerhans cells, should be susceptible to infection from both forms of the virus. And yet, previous work has shown that after sexual transmission, the T-tropic virus stays locked out of Langerhans cells. Researchers would like to know why this happens, saying the answer could provide important new clues for vaccine development or other HIV treatments.

In the December issue of Nature Medicine, a team of scientists report that they now have an answer.* They say that while in the skin--a site that closely reflects what happens on genital surfaces--Langerhans cells fail to produce a surface protein that the T-tropic virus needs to chemically unlock the cell membrane and come inside.

Just as significantly, they found that Langerhans cells produce adequate levels of another surface protein that the M-tropic virus uses, like a key in a lock, to enter these immune cells and infect them. "We hypothesized that this might be the case, but there was no direct evidence to support this hypothesis," said Andrew Blauvelt, M.D., an author on the paper and a scientist in the Dermatology Branch at the National Cancer Institute. "We now have that direct evidence."

Blauvelt said the findings of he and his colleagues at the Center for Biological Evaluation and Research at the Food and Drug Administration (FDA) build on recent work showing that HIV-1 enters immune cells using two distinct receptors. The M-tropic virus, which gets its name for its affinity for white blood cells called macrophages, anchors to the well-known CD4 protein that sits on the cell surface. But it also attaches to a second receptor called CCR5.

The T-tropic virus, which has an affinity for T cells and is associated with more advanced HIV infection, also anchors to CD4. But, it attaches to a different coreceptor on the cell surface, called CXCR4.

Marina Zaitseva, Ph.D., an FDA scientist and lead author on the paper, noted that Langerhans cells are capable of expressing both CCR5 and CXCR4, making it a potential target for both forms of the virus. But determining if it is a dual target had been problematic because previous work, for technical reasons, had focused on dendritic cells cultured from the blood, not the skin or genital surfaces. Langerhans cells are just one of several types of dendritic cells in the body.

"Dendritic cells from elsewhere in the body behave differently than those in the skin," said Hana Golding, Ph.D., an FDA scientist and senior author on the paper. "It's a little like trying to determine what happens at point A by looking at point X or Y. The comparisons can be misleading."

To get around this problem, Zaitseva and colleagues isolated Langerhans cells and macrophages, both of which are also suspected of initially interacting with HIV-1 on genital surfaces. Blauvelt said Langerhans cells from the skin act much like they do in the genital mucosa, making it possible to draw much closer correlations with receptor expression patterns on the genital surface than in the previous models.

The FDA scientists generated two polyclonal antibodies--one with specificity against CCR5 and the other targeting CXCR4. The idea being that the antibodies would recognize the individual receptors, allowing Zaitseva and colleagues to quantify levels of their expression on the cell surface.

The researchers found in 10 separate experiments that fresh Langerhans cells from the skin expressed the M-tropic co-receptor CCR5, at levels ranging in each experiment from 24 percent to 49 percent. In contrast, they found no indication that T-tropic receptors are present in the cell membrane.

These experiments raised another question. Would outer segments of the M and T-tropic virus, known as protein envelopes, also fuse to Langerhans cells in a similar pattern? If so, it would corroborate their previous antibody work and provide strong direct evidence that Langerhans cells can restrict the type of HIV-1 that it ferries to the lymph nodes, where the virus establishes its home base for further attacks on the immune system.

The researchers found that there was a correlation. Within three hours of exposure to the M-tropic envelopes, Langerhans cells expressing the CCR5 receptor began fusing together, called syncytia, a common characteristic of cells in culture that have been infected with HIV-1. The fusion continued over the duration of the 18-hour experiment. Meanwhile, Langerhans cells exposed to T-tropic envelopes failed to form syncytia, meaning the envelopes failed to fuse with them.

The researchers next turned to macrophages. Using macrophages derived from monocytes, or undifferentiated immune cells, they found high levels of both receptors expressed on the cell surface. But when they tested whether HIV-1 envelopes fused with the macrophages, the scientists found that fusion had occurred only for the M-tropic virus. Golding said she and her colleagues do not have an explanation for this, but they are now investigating the finding further.

Based on this month's paper, the scientists said it may be possible for researchers to focus on developing agents that block HIV-1 infection on the surface of immune cells, before the virus infects them. Golding pointed out that current drug therapy attacks HIV-1 inside immune cells, where it already has established an active infection. "Our data suggest that nature has provided a selective defense against some HIV-1 strains on the cell surface," said Golding. "It may be possible to begin thinking about how to develop a drug or vaccine that blocks these doorways from HIV-1 entry."

* The title of the study is, "Expression and Function of CCR5 and CXCR4 on Human Langerhans Cells and Macrophages: Implications for HIV Primary Infection." The authors are Marina Zaitseva, Andrew Blauvelt, Shirley Lee, Cheryl K. Lapham, Vera Klaus-Kovtun, Howard Mostowski, Jody Manischewitz, and Hana Golding.

For more information about cancer visit NCI's Website for patients, public and the mass media at http://rex.nci.nih.gov or NCI's main website at http://www.nci.nih.gov.