Cellular infection study partially explains lower HIV infection rates through oral sex

A study in which slices of human mucosal tissue were infected with HIV has partially explained why HIV is caught so much more easily through anal sex than through oral sex. The study, in which tissue samples from the tonsils and the rectum were infected with two different kinds of HIV, found that rectal CD4 cells express five times as much of the CCR5 receptor molecule on their surface as tonsil cells. This is the co-receptor for R5 or M-tropic virus, the variety of HIV that is usually transmitted. Rectal CD4 cells, once infected, also produced more of this virus than tonsil cells.

The study also found that in response to infection with the other kind of HIV, X4 or T-tropic virus, tonsil cells, but not rectal ones, secreted chemicals called chemokines that blocked further access to the CCR5 receptors.

However, while showing why CD4 cells in the tonsils are so much less susceptible to R5 infection than rectal ones, it does not show why these cells – most of which also carry the CXCR4 receptor – are so much less likely to be infected with X4 virus in vivo than in the laboratory. The researchers say that another so far undetected defence against X4 virus must exist.

The research team was led by scientists from the US National Institute of Child Health and Human Development in Bethesda, Maryland, and also included prominent microbicide researchers Ian McGowan from UCLA and Robin Shattock from St George’s Hospital, London.

In the experiment, so-called cellular explants were constructed by taking leftover tissue samples from rectal biopsies and tonsillectomies in HIV-negative patients. The rectal samples were rectosigmoid tissue taken from the junction of the rectum and colon 30cm from the anus. The explants consisted of slices of tissue mounted on gel rafts and kept alive in a culture medium. Both types of explants contained large numbers of CD4 and CD8 cells.

Within five hours of removal from the donor, the explants were exposed to two viral cultures containing strains of HIV called R5_SF162 and X4_LAL04.

The difference between R5 and X4 viruses is that they attach to two different cellular receptors, CCR5 which is found mainly on a proportion of the cells that patrol the mucosal surfaces of the body, and CXCR4 which is found on most T-lymphocytes.

Nearly all of the HIV that gets transmitted is R5 virus, though some viruses are ‘dual tropic’ and can use CXCR4 too.

Once within the body, HIV has a tendency to mutate into the X4 form, though even in patients with advanced disease no more than half the viral population is X4 or dual-tropic virus. X4 virus appears to be more virulent in that once it does infect cells it kills them more quickly and produces more HIV. However HIV seems to continue to prefer to infect cells via the R5 route.

The researchers used assays to:

• Detect how much HIV infectious cells were producing, using tests for both viral RNA and the viral p24 protein
• Determine what proportion of cells were carrying the CCR5 and CXCR4 receptors
• Detect a variety of immune-modulating proteins (cytokines and chemokines) produced by different kinds of cell.

Infected rectal cells produced more R5 virus than tonsil cells. Between days three and twelve of the experiment, tonsils cells infected with R5 virus produced only an eighth as much (12.4%) of the HIV p24 protein as cells infected with X4; rectal cells infected with R5 produced more than half as much (54%).

In terms of copies of HIV RNA, X4-infected tonsil cells produced, between days three and twelve, 45 million copies of RNA, whereas R5-infected cells produced three million copies. In contrast rectal cells infected with X4 produced about 28 million copies of RNA but cells infected with R5 virus 40 million copies.

X4 virus, as has been previously shown, is more cytopathic (destructive of cells) than R5 virus generally, but R5 infection in this experiment was more destructive of rectal CD4 than tonsillar CD4 cells. X4 infection killed off 80% of both tonsil and rectal CD4 cells whereas R5 virus killed off 4.6% of tonsil CD4 cells but 34% of rectal CD4 cells.

It has been established by other researchers that in acute HIV infection, a rapid wave of infection and destruction of CCR5-carrying gut lymphocytes occurs, and this pattern seemed to be repeating here.

About five times as many rectal CD4 cells as tonsil cells carried the CCR5 receptor, it was found. At the start of the experiment 15% of tonsil cells expressed the CCR5 receptor, 6% without expression of CXCR4. In contrast two-thirds of cells expressed CXCR4.

By day twelve of the experiment even fewer tonsil cells expressed CCR5, with the proportion of R5-expressing cells going down to 8.75%, 5.5% of them without CXCR4, whereas the proportion of CXCR4-expressing cells had slightly increased.

In contrast, in rectal cells at the start of the experiment 71% expressed CCR5, 31% exclusively, and 51% expressed CXCR4, only 11% exclusively. By day ten, the proportion of CCR5-expressing cells had gone down somewhat to 60% of cells, 12% expressing it without CXCR4, and CXCR4-expressing cells up to 84%, 36% exclusively.

Different CD4 cells infected with different viral strains expressed different levels of cellular defence and immunomodulating proteins. Rectal cells infected with both strains of virus produced high levels of certain cytokines that have little direct influence on HIV infection such as the growth factor GM-CSF. Tonsil cells infected with R5 virus showed little evidence of any significant cytokine/chemokine production.

However tonsil CD4 cells infected with X4 virus produced high levels of the chemokine chemicals MIP-1α, MIP-1β and RANTES (though in the case of RANTES the 2.5 fold increase in production was not statistically significant). CCR5 is the cellular receptor for these proteins, and a ‘chemokine blockade’ of cells could ensure that no further infection of R5-carrying cells happens under these conditions.

X4-infected tonsil cells also expressed higher levels of the chemokine SDF-1α. CXCR4 is the cellular receptor for this protein, although in the laboratory conditions of this study, high levels of it did not appear to inhibit ongoing X4 virus infection and production.

These findings provide a partial explanation for why HIV preferentially infects via the rectal rather than the oral route.

The researchers write: *In rectosigmoid tissue, R5 potential targets were much more abundant than in tonsillar tissue.”

They add: “The abundance of CCR5-positive CD4 T cells in rectosigmoid tissue makes the cytopathic impact of R5 [virus] more pronounced than in tonsillar tissue. In the latter R5 depletion is barely noticeable, whereas in rectosigmoid tissue more than 34% of CD4 T cells were depleted. These data may be relevant to the predominant mucosal transmission of R5 virus and the rapid depletion of GALT [gut-associated T lymphocytes] at the early stages of HIV-1 infection.”

The study shows why the rectum is susceptible to R5 virus and the mouth is barely susceptible. However it doesn’t explain why CXCR4-using virus is rarely transmitted. As these experiments show, even in rectal cells, in laboratory conditions X4 virus readily infects cells and when it does so they produce more virus.

Why doesn’t X4 virus get into cells when those cells are inside people? Increased SDF-1 secretion might be a partial answer, as might increased levels of a mysterious defence chemical called CD8 cell anti-HIV factor (CAF), whose mode of action has yet to be characterised; tonsil cells produced twice as much CAF as rectal cells. However these probably don’t provide the complete answer.

“Undoubtedly, there are additional mechanisms that serve as partial ‘gatekeepers’ restricting X4 infection in vivo,” comment the researchers.