LONDON - AIDS researchers have hypothesized for years that cytotoxic T lymphocytes must play an important role in the control of HIV infection, but no one has ever been able to prove that this is the case. Now a team of international scientists has provided evidence to support this theory.
The research confirms that the many strategies for producing a vaccine to protect against HIV infection by boosting the response of cytotoxic T cells specific for the virus are probably on the right tracks.
If cytotoxic T cells were effective in combating HIV, then people with many circulating cytotoxic T cells specific for the virus would be expected to have low levels of virus in their bloodstream, while those with few such cells would be expected to have high circulating levels of HIV.
Attempts to prove that this is the case have hitherto been hampered by the absence of a reliable method for quantifying cytotoxic T cells specific for particular antigens.
Using new technology, however, a team led by Andrew McMichael, professor of immunology at the Institute of Molecular Medicine of the University of Oxford, in the U.K., have now proved the truth of the hypothesis.
McMichael and his colleagues reported their results in Science, March 27, 1998, in a paper titled “Quantitation of HIV-1-specific cytotoxic T lymphocytes and plasma load of viral RNA.“
Working with scientists in Oxford and at the Aaron Diamond AIDS Research Center, in New York, McMichael's group used a recently developed reagent known as human leukocyte antigen-tetrameric complexes to identify cytotoxic T cells specific for HIV.
HIV-Specific T Cells Unmasked
All nucleated cells have human leukocyte antigen (HLA) molecules on their surfaces. These molecules present fragments of degraded proteins from inside the cell - including viral peptides if the cell is infected with a virus. If a cytotoxic T cell specific for HIV comes across a cell bearing HLA molecules containing fragments of HIV proteins, it will be stimulated to kill that cell.
The HLA-tetrameric complexes capitalize on this system. To make them, scientists express the genes for the component parts of the appropriate HLA molecule separately in bacteria. When, after purification, the two component HLA chains are mixed, together with the peptide and other components, they spontaneously recombine in the correct conformation.
Such tetramers will bind to those T cells specific for both the HLA molecule and the peptide of interest. They can be labeled with a fluorescent tag, which makes it possible to count them accurately with a cell sorter. By contrast, previously available methods have mainly relied on culturing cytotoxic T cells obtained from blood samples for several weeks, which can lead to various inaccuracies.
McMichael and his colleagues used the new tetramers to study blood samples taken from untreated HIV-infected individuals and stored frozen at the Aaron Diamond AIDS Research Center.
Graham Ogg, Medical Research Council clinical training fellow at the Institute of Molecular Medicine, and first author of the Science paper, told BioWorld International, “Once thawed, we added the HLA-tetrameric complexes to the white blood cells and were able to separate out those cytotoxic T cells specific for HIV. We also measured plasma RNA viral load in these samples. When we plotted these two, we found we had a very significant inverse correlation between them.
“Patients with high viral load had low levels of HIV-specific T cells and individuals with low viral load had high levels of T cells.“ This observation fits the original prediction, he added, that the cytotoxic T lymphocyte response is keeping the infection under control.
“This result,“ added Ogg, “gives people who are trying to develop a vaccine against HIV that will stimulate the cytotoxic T cell response some reassurance that they might be doing some benefit.“
The data presented in Science also support the widely held view that HIV is indeed a cytopathic virus - one which kills the cells that it infects fairly rapidly - rather than a noncytopathic virus, such as hepatitis B, which causes infected cells to produce virus in plenty, but without killing the cells.
McMichael and his colleagues compared levels of cytotoxic T lymphocytes specific for HIV with the rates at which the same individuals cleared the virus after starting triple-combination drug therapy.
If HIV were noncytopathic, cytotoxic T cells would be expected to play a large role in eliminating infected cells once drug treatment began. People with higher levels of cytotoxic T cells would be expected to clear virus more rapidly than those with lower levels.
In fact, the team found that there was no correlation between the number of cytotoxic T cells and the rate at which the virus was cleared after starting treatment. Ogg said, “This implies that the cytotoxic T cells must be killing the infected cells immediately before they would have been killed by the virus anyway. It means that cytotoxic T cells do not have an enormous effect on the half life of infected cells.“
Virus' Deadly Progression Targeted Next
Nevertheless, Ogg and his co-authors point out in Science, that small change in the life span of productively infected cells may have a large effect on viral burst size. This would explain how cytotoxic T cells exert their effect in controlling viral load.
Next, Ogg and his colleagues plan to use HLA-tetrameric complexes to study people at different stages of infection with HIV, in order to characterize the nature of the immune response to HIV and to determine which parts of the immune response would need to be stimulated in order to develop an effective vaccine.
Ogg said, “Colleagues in Andrew McMichael's lab are working on developing HIV vaccines and collaborating with other groups around the world, using tetramers to look at the response to vaccination. The ultimate goal is to make a vaccine or have some strategy where, when people are treated with drug treatment, they may also benefit from boosting their immune system as well.“ *