BioWorld International Correspondent
LONDON - A previously unknown way in which cells generate protein fragments for recognition by the immune system has been identified by researchers working in Germany, France and Israel. The discovery has important implications for the design of vaccines to protect against viral infections such as HIV, the team predicted.
Peter-Michael Kloetzel, who led the research, told BioWorld International, "This could open up an entirely new way of thinking about how peptide fragments are generated inside cells and about how the entire protein-breakdown machinery of the cell works."
Kloetzel, professor of biochemistry at the Institut fur Biochemie-Charite at the medical school of the Humboldt-University in Berlin, and his collaborators report their findings in an article in Nature Immunology (advance online publication Feb. 24, 2003) titled: "An essential role for tripeptidyl peptidase in the generation of an MHC class I epitope."
The first step in mounting an immune response against a viral infection involves infected cells breaking down viral proteins into much shorter fragments called peptides. The cell has to trim these to exactly the right length and in the correct format to fit the groove of major histocompatibility complex (MHC) class I molecules.
These smaller fragments, known as epitopes, appear on the cell's surface, held in the MHC class I molecules. The infected cell presents them to cells, such as cytotoxic T cells, that can kill infected cells, as well as trigger a wider immune response against the virus. Until the study by Kloetzel and his group, the scientific community believed that production of epitopes occurred solely at a large intracellular complex of proteins known as the proteasome.
Study of how the proteasome processes viral proteins is crucial to the development of new vaccines. In the search for a vaccine to protect against HIV, many researchers have studied the epitopes of the Nef protein of HIV.
This protein is one of the first to be synthesized by the cell once it has become infected, and it is capable of stimulating a strong immune response. It could therefore be a good component of a vaccine against HIV because it could trigger an immune response before viral structural proteins begin to be manufactured.
Kloetzel and his collaborators knew that a particular epitope of HIV nef, comprising amino acids 73 to 82, appeared only in infected people who were HLA-A3 or HLA-A11. Researchers had observed that adding an inhibitor of proteasomes to cultures of infected cells from such people failed to stop production of this epitope. They theorized that perhaps epitopes like this one, which have a lysine residue at their C-terminal end (the end that anchors it to the MHC class I molecule), were processed less efficiently by proteasomes.
Furthermore, researchers also knew that people infected with certain isolates of HIV were unable to present this same HIV nef epitope, and that sequencing studies had shown that these isolates had a mutation flanking the C-terminal lysine residue.
The group set out to examine the reasons for these observations in more detail. An initial study showed that purified proteasomes were unable to generate the HIV nef epitope that they were interested in. To make the conditions more similar to those in vivo, the researchers even fused the entire nef protein to a protein that was known to be processed by the proteasome - but still they could not obtain the epitope in question.
Kloetzel told BioWorld International, "This left us completely frustrated for six months because we did not know what to do next." Eventually, they decided to see what happened if they added specific proteasome inhibitors to dendritic cells expressing full-length nef. They chose these cells because they are the category of cells mainly responsible for inducing an immune response during HIV infection.
"By this stage," Kloetzel said, "we were no longer surprised to find that proteasome inhibitors did not impair the presentation of the epitope in this system. So we went on to test many different protease inhibitors, to see what happened."
They were in luck. An inhibitor described as specific for a protease called tripeptidyl peptidase II (TPPII) completely abolished the presentation of the epitope.
When they repeated their first set of experiments and added purified TPPII, they found that, this time, they were able to obtain the epitope.
What are the consequences of this finding for vaccine design? "This epitope would normally have been included in a polyepitope vaccine, on the assumption that the proteasome system would take care of it," Kloetzel said. "Now it appears that it would not be appropriate to include it. This also raises the question of whether you could design a specific vaccine that relies more on the function of TPPII to generate a more specific response against this epitope."
He and his colleagues are now embarking on studies to find out how general this mechanism is, how many other epitopes also are processed in this way, and whether TPPII plays a specific role in dendritic cells.