The identification of the disulfide bonds in HIV gp120 whose reduction is required for cell entry and their manipulation for immunogen design

Infection by the Human immunodeficiency virus (HIV) is the cause of acquired immunodeficiency syndrome (AIDS) and is a major cause of death worldwide. Despite extensive research efforts the search for an effective vaccine has not yet been successful and there is a continued need for new candidates to be assessed. The virus enters the cell via interaction between the virus envelope glycoprotein and the target cell surface, in particular two cell surface molecules called CD4 and CCR. Following contact between these molecules a number of biochemical steps occur which result in the virus penetrating the cell to start the infection. The precise nature of the biochemical steps required is still a matter of research but if they are identified clearly they may be useful in the design of the new potential vaccines. We have discovered one particular biochemical change, disulfide bond reduction, which appears to be important for virus entry. If this step is inhibited virus infection cannot occur suggesting it is a key intermediate stage in the entry process. We want to use a new technology involving very sensitive mass spectrometry to identify the particular disulfide bonds of the virus envelope glycoprotein that are reduced during the infection process in order to provide a greater level of clarity about the entry process than is currently the case. When the particular disulfide bonds are known we will make some of the reduced protein and compare its properties to the normal, unreduced form. We will also test if this intermediate protein is a key part of the HIV infection process by using it to generate antibodies which will be tested to see if they prevent infection. Antibodies, the host's response to infection, which bind to the reduced protein and stop its function will prove that the reduced protein can be considered a valid vaccine candidate.

There is ample evidence in support of the fact that HIV gp120 undergoes reduction at the cell surface concomitant with its action, together with gp41, in viral entry. Reduction affects at least 2 disulfide bonds, occurs after CD4 binding and is required before fusion can occur. As such it is a key intermediate stage in the entry process. However, the exact identity of the reduced intermediates, that is, which cysteines are reduced and exactly where and when they arise, is not known. We have developed an assay system that is able to identify reduced Cys residues in a complex of gp120/CD4 /reductant and we plan to identify the gp120 disulfide bonds that are reduced during cell entry and confirm the cell surface isomerase responsible for cleavage. We suggest that, as key intermediates, partially reduced gp120s may represent novel immunogens able to generate antibodies that access and block the fusion reaction. We will test this hypothesis by immunisation of small mammals and assessing the sera generated for breadth of antibody response and for the presence of neutralising antibodies. Our work will contribute new information to models of the mode of action of HIV Env and test rationally chosen gp120 variants as novel vaccine candidates.

Our work has the potential to impact many people in many areas of society, nationally and internationally. It will impact on people suffering from HIV infection by describing advances that may lead to improve therapies or by providing those improved therapies directly. As up to 33 million people carry the virus representing an extraordinary possible outreach. Our work will benefit many groups in the pharmaceutical industry concerned particularly with the development of improved candidate vaccines for the treatment of human immunodeficiency virus infection. A further immediate beneficiary in the pharmaceutical sector will be those groups involved in the isolation of small molecular weight compounds as drugs that inhibit the HIV entry reaction. Our target molecules are host encoded and occur during virus entry and in this regard they may be considered similar to other cellular entry targets such as CCR5 for which a very effective drug, Maraviroc, has been successfully developed and to which drug resistance is minimal. The pharmaceutical sector beneficiaries are to be found within the UK, Europe and worldwide. In the event that our data provide key information for the generation of novel vaccines or antivirals, the market is considerable and profits from the pharmaceutical industry will benefit their shareholders, including pension funds, and allow increased support of philanthropic activities, for example the development of drugs for neglected diseases. Successful drug development based on our work will benefit the NHS through a reduction in bed residence and associated nursing and treatment costs. Our work will benefit education, particularly of schoolchildren, by providing a clear demonstration of the value of science based drug and vaccine development. The existing interest in vaccines and HIV will be augmented and will increase the public understanding of science, promoting the sciences as a valuable study for children in school and in further education.