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Dr. Binley's lab is interested in developing an effective vaccine for HIV-1. In particular, he is interested in finding a vaccine that can induce minute virus-fighting molecules in the blood, called neutralizing antibodies. The history of successful vaccines against other infectious agents suggests that such antibodies may well be essential. One problem that vaccine researchers have faced is one of authenticity. For neutralizing antibodies to kill the virus, they must bind to the form of the coat protein that allows the virus to infect human cells. However, HIV cleverly present other decoy forms of its coat protein that appear to distract the immune responses, so that the virus can operate "under a veil".
Dr. Binley's lab is focused on making a vaccine that eliminates all the decoy forms of the coat protein, so that immune responses to this modified vaccine are focused squarely on the most relevant form of the coat protein. Aside from this core work on vaccine design, Dr. Binley's lab participates in a Consortium of HIV researchers whose job it is to monitor the strength and specificity of antibody responses to vaccine candidates. This group also looks at how certain HIV-1-infected people manage to generate broadly reactive virus fighting antibodies. A better understanding of such responses might help us to develop vaccines that can induce similar immunity.
A vaccine represents a cheap and practical option for controlling the global HIV epidemic and as such is one of the most compelling challenges in biomedical research today.
In the last 25 years since the discovery of HIV, attempts to induce "neutralizing antibodies" that are able to block HIV infection has been the single biggest challenge in vaccine development. Traditional approaches that have worked for viruses like smallpox, polio, and measles have so far failed for HIV. It is sobering to consider that only a handful of effective neutralizing antibodies (from among many hundreds) have been identified to date. It is becoming increasingly clear that the vast majority of antibodies generated during HIV infection are only able to recognize defective or "junk" forms of the HIV Envelope (Env) coat proteins. To make progress, we may need to understand why so many of these "junk" antibodies are induced by current HIV vaccine candidates.
We are using virus-like particles (termed "VLPs") - also known as pseudovirions - as a central commodity to investigate a variety of questions related to Env conformation, function, immunogenicity, and neutralization. VLPs are synthetic viruses that look exactly like the real HIV virus, except that they are modified and treated to make them safe and non-infectious. Our approaches using VLPs stem from the fact that these particles are a facile way of analyzing the functional form of Env: gp120/gp41 trimers (Fig. 1).
Figure 1: Electron micrograph of a HIV-VLP particle. The surface of the particle is stippled with Envelope proteins that resemble the predicted trimeric structure (photograph, courtesy K. Roux; inset, courtesy P.Kwong).
Over the last few years, we have developed a number of protocols involving VLPs (outlined in Fig. 2). VLPs can be used to immunize animals with a view to inducing neutralizing antibodies. VLPs can also be used to investigate antibody neutralization, and modified assays can determine neutralization mechanism. In addition, VLPs can be used in virus capture, native PAGE, and ELISA assays, useful in mapping anti-Env antibodies and for evaluating the conformation of Env on particle surfaces. The common use of VLPs in these methods facilitates a direct cross-referencing of data, empowering each assay.
Figure 2: Multiple uses of VLPs. VLPs with or without modified forms of Env on their surfaces can be used to try to induce neutralizing antibodies in animals, to analyze neutralization and its mechanism, to map binding and neutralizing antibodies to HIV-1, and to investigate the conformation of Env on particle surfaces.
i) VLPs as a platform for an HIV vaccine
A VLP approach to a HIV vaccine is exciting for several reasons. First, from the most elementary perspective, making a vaccine that looks like the real virus makes perfect sense if one wishes to protect against a live HIV virus challenge. Second, it is a relatively unexplored approach that can be easily adapted or modified by simple molecular biology techniques, meaning that many exciting possibilities remain untested. Functional trimers (Fig.1) are exclusively recognized by neutralizing antibodies and represent ideal target molecules for a vaccine. However, unmodified particles (wild type, WT-VLPs) have a tendency to shed gp120 (Fig. 3A), leaving behind nonfunctional Env that may have a negative effect on the ability of the particles to induce neutralizing antibodies. One approach to eliminate this problem is to ablate the cleavage site in the gp160 Env precursor (UNC-VLPs, Fig. 3B) or to introduce a disulfide bond to link gp120 and gp41 subunits in fully cleaved trimers (SOS-VLPs, Fig. 3C). Recently, we have made significant advances toward eliminating all “junk” forms of Env from particles. Thus, we are now positioned to test pure native Env trimers as immunogens for the first time.
Figure 3: Shown on the cover of Virology, September 30, 2007, a schematic depiction of VLP candidate vaccines. A) unmodified wild type (WT-VLPs) bearing functional gp120/gp41 trimers. Gp120 can sometimes be shed from these trimers, leaving behind gp41 stumps. B) UNC-VLPs in which the gp120-gp41 cleavage site is ablated to eliminate gp120 shedding, but which renders trimers non-functional. C) SOS-VLPs in which a disulfide bond links gp120-gp41 to prevent shedding, while retaining functional properties similar to WT-VLPs
ii) Studying Env conformation and mapping the specificity and mechanism of neutralization.
Some VLPs are capable of a single round of infection of susceptible cells. Infection is simple to assay using a luciferase readout. A powerful aspect of VLPs is that since they are expressed by plasmid transfection, the researcher has the ability to express viruses bearing any Envelope of choice (e.g. from North America or Africa, from a drug resistant mutant or field isolate, from HIV or its simian counterpart, SIV). Because Env is expressed in a functionally relevant form, VLPs provide a way to examine Env structure-function relationships, and to investigate Env recognition by neutralizing and non-neutralizing antibodies. In particular, we are using VLPs to understand better why some HIV+ patient plasmas broadly neutralize HIV-1, while others do not. We have developed a series of assays (Fig. 2) including:
Modified neutralization assays. We have developed several new formats to help identify the stage at which an antibody can neutralize virus, i.e. pre-attachment, post-CD4 binding, post-CD4/CCR5 binding. For example, neutralization post-CD4/CCR5 binding can be assayed by allowing SOS-VLPs (Fig. 3) to attach to target cells, engaging CD4 and CCR5. The SOS bond prevents infection from proceeding until a low concentration of reducing agent is added to disrupt the disulfide bond between gp120 and gp41. Thus, neutralizing antibody can be titrated against the cells with receptor-bound virus and then reducing agent added to allow any residual virus to infect (the non-neutralized fraction).
Virus capture assays. Here, an antibody, usually monoclonal, is used to capture virus on an ELISA plate, by a protocol very similar to traditional ELISA. Susceptible cells are then added, to evaluate how efficiently the virus was captured. Surprisingly, several groups have now confirmed that many non-neutralizing "junk" antibodies are able to specifically capture virus, suggesting non-functional Env as well as functional trimers are present on virus surfaces. We have adapted virus capture for mapping antibody specificities. This works by titrating the antibody sample against the VLPs and looking for a decrease in virus capture by any of a panel of index monoclonal antibody prototypes directed to common Env epitopes.
Blue native PAGE (BN-PAGE). Env trimers derived from particles can be visualized in native gels. We have shown that only neutralizing monoclonal antibodies and sera are able to bind to these trimers, retarding their migration in native conditions. We are now adapting this method to try to map the important contact residues of neutralizing antibodies in neutralizing patient plasmas and vaccine sera. We are also using this method to investigate the conformation of various modified forms of Env expressed on VLP surfaces.
Together, these assays can be used to profile neutralizing antibodies in complex polyclonal samples and to help define the properties of various HIV Envs and mutants, with an overarching view to advancing HIV vaccine research.
Honors & Awards
Selected Publications (10 of 62)