Group Trkola

HIV Entry
Dissecting the Mode of Action of HIV-1 Entry Inhibitors and Neutralizing Antibodies
Defining novel inhibitors of the HIV entry process
Viral fitness
Humoral immunity in HIV infection
In vivo and in vitro analysis of the neutralizing antibody response to HIV-1
The role of antibody dependent cellular cytotoxicity (ADCC) in HIV infection
HIV escape to neutralizing antibodies and entry inhibitors
The B cell response to HIV at different disease stages

HIV Entry

Extensive studies over recent years have brought us a profound knowledge of the entry process of HIV and have identified in this process several targets for drug interference. In order for HIV to infect a cell, the virus requires expression of CD4, the primary receptor for HIV-1, and a fusion coreceptor, most commonly the chemokine receptors CCR5 and CXCR4. The entry process proceeds via a cascade of events that provide multiple opportunities for therapeutic intervention, and several agents targeting this process have been developed over recent years. Three essential steps in the entry process have been defined: (i) the attachment to the host cell and binding of the gp120 envelope molecule to the primary receptor CD4, (ii) conformational changes allowing interaction with the coreceptor, and subsequent induction of (iii) the actual fusion step between viral and host membrane

Dissecting the Mode of Action of HIV-1 Entry Inhibitors and Neutralizing Antibodies

Livia Berlinger, Claudia Ruprecht, Peter Rusert, Alexandra Trkola

Attachment of the virus to target cells, the interaction with its cellular receptors and the following events leading to fusion and entry are major steps in the viral life cycle. Hence, both prophylactic and therapeutic interventions are sought for that interfere with this process. Neutralizing antibodies target specifically HIV entry and are in the focus of ongoing vaccine development. Although, much has been learned on the HIV entry process and neutralizing antibody action over the past 20 years, the precise mode of action of the various classes of neutralizing antibodies (NAbs) and entry inhibitors known date have not been completely unraveled. In this project we aim to dissect the influence of NAbs and inhibitors on attachment, receptor engagement and fusion. By defining their mode of action, we hope to get valuable insight on their target specificity and in turn obtain useful knowledge aiding vaccine development.

Defining novel inhibitors of the HIV entry process

Anders Krarup, Axel Mann, Peter Rusert, Alexandra Trkola

In collaboration with Prof A. Plückthun, Department of Biochemistry, University of Zürich and Melissa Robbiani, Population Council New York

Considerable effort has been put into investigating the interaction of the virus with its entry receptors and the identification of potential antiretrovirals. Several types of entry inhibitors have been developed that block either the interaction of the virus envelope with the primary receptor CD4, a coreceptor, or the fusion reaction. However, thus far only two entry inhibitors have been licensed for clinical use, and a wider spectrum of entry inhibitors for application in treatment and prevention is urgently needed. Together with our collaborator Andreas Plückthun and his team at the Depeartmenet of Biochemistry at the University Zürich, we are aiming to develop novel inhibitors of the HIV entry process using designed ankyrin repeat protein (DARPin) libraries. DARPins are attractive candidates for protein-based inhibitors as they share many features of antibodies while having a higher chemical and physical stability and displaying a different binding mode compared to antibodies (Binz HK et al Nature Biotechnology 2005). In a previous study together with our collaborators at Molecular Partners Zürich, the co-inventors of this technology, we have already successfully used this technology to derive DARPins with high affinity to CD4. These CD4 binding DARPins showed potent and specific inhibition of HIV entry (Schweizer et al. PLoS Pathogens 2008). Their potential as an ingredient of an effective microbicide is currently under investigation.
In the present project we are applying this novel technology for the selection of specific binders against the HIV envelope glycoproteins gp120 and gp41 with the aim to derive agents with antiviral activity for use as microbicides.

Viral fitness

Peter Rusert, Therese Uhr, Alexandra Trkola

In collaboration with the groups of Huldrych Günthard and Marek Fischer at the Division of Infectious Diseases,University Hospital Zurich

Biological properties of HIV-1, namely tropism, cytopathicity and replication rates are relevant parameters in AIDS pathogenesis. It is generally believed that in the course of the infection the virus increases its fitness. As an example, the switch in coreceptor usage from CCR5 to CXCR4, which occurs in approximately 50% of patients, is associated with a more vigorous viral replication and a rapid disease progression. Viral fitness reflects the aptitude of a viral isolate to replicate in a given host system and is a consequence of the capacity of the virus to efficiently enter and infect target cells and to establish and spread the infection. The efficacy of this process is further influenced by the availability of target cells, adaptive and innate immune responses, genetic host factors and antivirals. Our research in this area focuses on the evolution of the viral envelope proteins gp120 and gp41 during the disease course and the impact genetic shifts in these proteins have on viral tropism, fitness and sensitivity to entry inhibitor.

Humoral immunity in HIV infection

Humoral immunity in concert with cellular immune responses is thought to be required for natural and vaccine-induced control of HIV-1 infection. Efforts to generate vaccines based on humoral immunity are underway but have failed so far to elicit immune responses that are comparable in breadth and potency to those elicited during natural infection. The success of preventive and therapeutic vaccination will greatly depend on how capable immunogens are in eliciting broad immune responses, how these can be maintained and, most importantly, how effective the evoked responses are in suppressing viremia. The overall aim of our research is to gain a better understanding of the humoral responses elicited to natural HIV infection in order to provide new insights for future vaccination strategies.

In vivo and in vitro analysis of the neutralizing antibody response to HIV-1

Peter Rusert, Claudia Ruprecht, Irene Abela, Livia Berlinger, Alexandra Trkola

In collaboration with the groups of Huldrych Günthard and Marek Fischer at the Division of Infectious Diseases, University Hospital Zurich

Neutralizing antibodies against HIV-1 are directed against specific epitopes on the envelope glycoproteins gp120 and gp41, and inhibit viral entry by blocking virion attachment to its receptors or membrane fusion. During natural infection the effect of the autologous neutralization response appears to be limited since the virus rapidly escapes the immune pressure in most individuals. Nevertheless, rare potent monoclonal antibodies with broader activity have been isolated from infected individuals. Until recently the activity of neutralizing antibodies in infected humans remained circumstantial and was best evidenced by the rapid selection of neutralizing antibody escape variants. Direct proof of the activity of neutralizing antibodies in established human HIV infection was provided in a recent passive immunization trial from our group (Trkola et al. Nature Medicine 2005). The current main effort of our research activities is directed towards studying the humoral immunity to HIV-1 in distinct, well defined patient groups to get a better understanding of the magnitude, impact and timing of the neutralization response. Rare human monoclonal antibodies with potent neutralizing activity have been isolated but antibodies with similar specificities appear to be uncommon in vivo and have proven to be difficult to be induced by vaccination. Despite the fact that patients who mount a high neutralization response have been identified, it still remains largely unclear which epitopes neutralizing antibodies in the sera of these patients recognize. A primary goal of our research is therefore to define epitopes on the HIV envelope proteins that elicit potent neutralizing activities in vivo.

The role of antibody dependent cellular cytotoxicity (ADCC) in HIV infection

Lucy Reynell, Claudia Ruprecht, Alexandra Trkola

In collaboration with the group of Huldrych Günthard at the Division of Infectious Diseases, University Hospital Zurich.

Antibodies bound to viral proteins on the surface of infected cells, can, via their constant regions, recruit Fc receptor bearing cells, such as NK cells. Fc receptor cross-linking by antigen bound IgG initiates destruction of virally infected cells in a process termed antibody dependent cellular cytotoxicity (ADCC). Despite detection of ADCC mediating antibodies in patient sera, the impact of ADCC in HIV viremia is currently unclear.
One major obstacle in studying ADCC of HIV infected cells has been the lack of suitable assay systems to quantify autologous ADCC. In order to reveal the capacity of an individual patient’s antibody repertoire to initiate ADCC against cells infected with their own viral strain, we aim to develop a novel FACS based assay. To characterize the ADCC mediating potential of individual antibodies we will standardize the assay for various parameters including target cell antigen expression and cytotoxic cell input. The latter is particularly important as the cytotoxic activity of natural killer cells varies considerably between donors.

HIV escape to neutralizing antibodies and entry inhibitors

Peter Rusert, Claudia Ruprecht, Alexandra Trkola

In collaboration with the group of Huldrych Günthard at the Division of Infectious Diseases, University Hospital Zurich.and Polymun Scientific, Vienna.

The neutralizing antibody response to HIV-1 is confronted with continual viral escape, which will also affect efficacy of future vaccines. Thus, a profound knowledge of escape pathways both on the host and the virus level is pivotal for deciphering the subsequent consequences for the patient. This project investigates the development of viral escape mutants in vitro and in vivo and aims to define how the humoral immune system adapts to the newly emerging variants. The research builds on our extensive experience with the neutralizing antibodies 2G12, 2F5 and 4E10, which we use as models to define determinants that drive the evolution of viral escape. A particular focus is on studying the development of the neutralizing antibody response to viral escape mutants. While it has been shown that the virus rapidly escapes the neutralizing antibody response in vivo, it is presently not known how the immune system in turn reacts to the newly emerging virions.

The B cell response to HIV at different disease stages

Claudia Ruprecht, Alexandra Trkola

The overall aim of this project is to explore how and where antibodies to HIV-1 antigens are induced at different disease stages, what the underlying mechanisms of antibody induction to specific viral antigens are, and what we can learn from antibody induction during natural infection for vaccine design. Unraveling the specifics of antibody induction is of central importance for the development of effective vaccination strategies. Specifically we investigate the individual and general requirements of viral antigens in terms of quantity, antigen presentation, length of exposure to antigen, T cell help and duration of the responses. A definition at which disease stages novel antibody induction can be achieved will allow us to determine when therapeutic vaccination based on humoral immunity can be successful.