Group Trkola

Group Trkola 2015

Group members

List of publications (NCBI)

List of publications (ResearchID)

Our group focuses on two central topics: The HIV entry process and the humoral immune response elicited during HIV infection.

HIV Entry - Introduction

Extensive studies in recent years have brought us a profound knowledge of the entry process of HIV and have identified several targets for drug interference. In order for HIV to infect the virus requires target cells which express CD4, the primary entry receptor, and a coreceptor, most commonly the chemokine receptors CCR5 or CXCR4. Three essential steps in the entry process have been defined: (i) 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 (iii) subsequent induction of the actual fusion step between viral and host membrane.

This cascade of events the entry process goes through provides multiple opportunities for therapeutic intervention. Most importantly, HIV infection elicits a humoral immune response which gives rise to envelope directed neutralizing antibodies that have the capacity to block the entry process. While in principal effective, HIV has evolved intricate measures to prevent and escape attack by neutralizing antibodies. This hinders successful immune control of the virus in infected individuals and renders vaccine development extremely challenging.

Our group is interested in studying molecular details of the HIV entry process, its stoichiometry, its role in shaping viral fitness, envelope determinants which influence transmission from host to host and the spread within, the evolution of the envelope genes during pathogenesis, inhibition by neutralizing antibodies elicited in the course of the infection and the design and function of entry inhibitors. A specific interest of our research is dedicated towards understanding viral evasion of neutralizing antibodies and entry inhibitors.

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

Oliver Brandenberg, Peter Rusert

Neutralization of HIV by antibodies is generally attributed to antibody occupancy of the envelope trimers and interference with viral attachment to host cell receptors or entry, but the precise underlying molecular mechanisms leading to neutralization by most neutralizing antibodies identified to date await clarification. In the frame of this project we aim to dissect the influence of neutralizing antibodies and entry inhibitors on virus attachment to target cells, receptor engagement and fusion. By defining the mode of action of a diverse set of entry inhibitors and neutralizing antibodies, we hope to gain valuable insight on their target specificity and in turn obtain useful knowledge aiding vaccine and entry inhibitor development.

For a recent publication on this topic by our group see:

C. Ruprecht et al J Exp Med 2011

Stoichiometry of HIV Entry and Neutralization

Oliver Brandenberg, Carsten Magnus, Peter Rusert
In collaboration with Roland Regoes; ETHZ

Stoichiometric aspects of HIV entry and neutralization – that is, how many trimers, target cell receptors or antibodies are involved in the entry and neutralization processes – are currently not known. Within this project we attempt to resolve key components of the molecular interplay to resolve the stoichiometries of HIV-1 and HIV-2 entry and neutralization. We further aim to unravel whether or not differences in the stoichiometry of entry between free virus and cell-cell transmission of HIV exist. (see also section below and Abela et al., PLoS Path 2012). Overall we aim with this project to gain insight into the molecular basis of HIV-1 and HIV-2 entry, transmission and its inhibition by antibodies and thereby improve our understanding of virus pathogenesis and to provide directives for entry inhibitor and antibody based vaccine design.

For recent research from our group members on this topic see:

Magnus et al., J Virol 2009

Magnus and Regoes, PLoS Comput Biol 2010

Magnus and Regoes PLoS ONE 2012

Rusert et al J Exp. Med 2011

Abela et al., PLoS Path 2012

The role of the V1V2 domain in gp120 function and shielding

Oliver Brandenberg, Jacqueline Weber, Carsten Magnus, Peter Rusert

The variable loop domains 1 and 2 (V1 and V2) of the gp120 envelope glycoprotein are known to play an important role both in HIV transmission and in shielding against neutralizing antibodies, yet the underlying mechanisms of V1V2 involvement in entry and evasion from neutralization are not completely understood. The V1V2 loop can both function as a direct target for neutralizing antibodies elicited in vivo and shield distant neutralization-sensitive domains on the viral envelope such as the V3 loop, CD4 induced (CD4i) epitopes and the CD4 binding site (CD4bs). Understanding how the V1V2 domain steers neutralization sensitivity is thus of pivotal importance to allow vaccine induced antibody responses to be tailored to counteract its effect. How V1V2 shielding is provided for in the context of the native envelope spike is currently not conclusively resolved as we lack information on the precise spatial arrangement of the envelope trimer and in particular the inter-subunit contact areas as structural information on the variable loops within HIV-1 gp120 is still scarce and in the case of the V1V2 loop missing. It has however been ascertained that the V1V2 and V3 domains are positioned in the apex of the viral spike and that therefore a direct interaction between the loops is highly likely. We recently conducted a study to investigate whether inter- or intra-subunit contact between the V1V2 and V3 loop is the main mode of V1V2 shielding of the V3 loop. Our results strongly support a model where the V1V2 domains protect the V3 loop on neighboring (adjacent) gp120 subunits within the envelope trimer. In continuing our research on the V1V2 domain we currently investigate its role in the entry process, transmission and evolution during disease progression.

For recent research from our group members on this topic see:

Rusert et al J Exp. Med 2011

The role of cell-to-cell transmission in HIV infection

Lucia Reh, Oliver Brandenberg, Peter Rusert

HIV is known to spread efficiently both in a cell-free state and from cell to cell, however the relative importance of the cell-cell transmission mode in natural infection has not yet been resolved. Likewise, it remains to be determined to what extent cell-cell transmission is vulnerable to inhibition by neutralizing antibodies and entry inhibitors. We recently reported on neutralizing antibody activity during cell-cell transmission using specifically tailored experimental strategies which enable unambiguous discrimination between the two transmission routes. We demonstrated that the activity of neutralizing monoclonal antibodies and entry inhibitors during cell-cell transmission varies depending on their mode of action. While gp41 directed agents remain active, CD4 binding site (CD4bs) directed inhibitors, including the potent neutralizing mAb VRC01, dramatically lose potency during cell-cell transmission. This implies that CD4bs directed mAbs act preferentially through blocking free virus transmission, while still allowing HIV to spread through cell-cell contacts. Thus providing a plausible explanation for how HIV maintains infectivity and rapidly escapes potent and broadly active CD4bs directed antibody responses in vivo.

We currently investigate to what extent the neutralizing antibody response elicited during HIV infection is capable of blocking cell-cell transmission and at what disease stages such responses evolve. A further emphasis of our work is dedicated towards unravelling the molecular details of cell-cell transmission and the ensuing evasion from inhibitors.

For recent research from our group members on this topic see:

Abela et al., PLoS Path 2012

Novel inhibitors of the HIV entry process

Axel Mann, Nikolas Friedrich, Emanuel Stiegler, Jacqueline Weber, Therese Uhr, Peter Rusert
In collaboration with the groups of Prof A. Plückthun, Department of Biochemistry, UZH, Prof J. Robinson, Institute of Organic Chemistry, UZH and Melissa Robbiani, Center for Biomedical Research, Population Council, New York City

Considerable effort has been put into investigating the interaction of the virus with its entry receptors and the identification of inhibitors of the entry process. 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. In the frame of this project we utilize the Designed Ankyrin Repeat Protein (DARPin) technology developed by the group of Prof. A. Plückthun to isolate novel inhibitors of the HIV entry process. DARPins are attractive candidates for protein-based inhibition 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 the present project we are applying the DARPin 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.

For recent research from our group members on this topic see:

Schweizer et al. PLoS Pathogens 2008

Pugach P et al PLoS one, 2010

Gp120 Shedding

David Beauparlant, Oliver Brandenberg, Peter Rusert

Neutralizing antibodies can induce conformational changes in the envelope trimer by binding to their cognate epitopes which can lead to subunit dissociation, i.e. release of gp120 monomers from the trimer. This process is called gp120 shedding. We and others have demonstrated that a variety of neutralizing antibodies as well as CD4 have the capacity to induce gp120 shedding. We recently demonstrated that antibodies specific to the Membrane-Proximal External Region (MPER) of gp41 (2F5, 4E10) are inducing gp120 shedding. This process is tightly linked to the neutralizing action of these antibodies and leads to irreversible neutralization of HIV (Ruprecht et al. JEM, 2011). In the frame of this project we are currently investigating the capacity of a broad range of neutralizing antibodies to induce shedding as well as the sensitivity of virus strains at different disease stages to antibody and CD4 induced gp120 shedding.

For recent research from our group members on this topic see:

Ruprecht et al. JEM, 2011

Viral fitness

David Beauparlant, Jacqueline Weber, Therese Uhr, Peter Rusert
In collaboration with the group of Huldrych Günthard, Division of Infectious Diseases, University Hospital Zurich

Biological properties of HIV-1, namely tropism, cytopathicity and replication rates are considered relevant parameters in pathogenesis of HIV induced disease. 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 as well as to establish and spread the infection. Overall virus fitness in a host is further influenced by the availability of target cells, adaptive and innate immune responses, genetic host factors and antiviral drug treatment. 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 features such as viral tropism, envelope in vitro fitness and stability of the envelope glycoprotein trimer.

Mathematical modeling of virological phenomena

Carsten Magnus

Besides experimental techniques, mathematical models become more and more important to address biological question. In general, mathematical models can be used for (i) parameter estimation, (ii) comparing hypothesis, and (iii) for making predictions in specific settings. We combine all three approaches with experimentally obtained data to gain more inside into the following questions:

How many HIV spikes have to bind and which requirements must the spike fulfil for viral entry?

Spikes on the viral surface can establish contact to target cell receptors, finally resulting in infection of the cell. The number of spikes needed to bind to cell receptors for infection, the stoichiometry of entry, is not known. Experiments with pseudotyped virions expressing mixed trimers are employed to study the stoichiometry of entry. However, these experiments contain high levels of stochasticity and mathematical models help to interpret the data.

HIV spikes consist of three identical subunits. We are also interested in the requirements of the subunits within a spike to successfully take part in viral entry, the subunit stoichiometries.

How many antibodies need to bind to an HIV virion for neutralization?

This question can be broken down to the study of the numerical requirements for viral entry (see above) and the number of antibodies rendering one viral spike non-functional, the stoichiometry of trimer neutralization. When scaling these parameters up to the virion level one has to consider unproductive binding, i.e. binding of an antibody without any neutralization effect.

How does HIV evade antibody neutralization?

The V1V2-loop plays a very prominent role in shielding epitopes against antibody binding. It is still under debate which subunit is protected by this structure. Mathematical models help to predict the outcomes of experiments and tailor these experiments for most significant results.

How can an infection with HIV be modelled and what do these models predict?

Since the early 1990s mathematical models have been employed to tackle different aspects of HIV infection. These models can be used to infer important parameters of an infection such as the virion clearance rate or the lifespan of infected cells. In addition, these models can be used to make predictions on the progression of the disease and the effects of treatment strategies.

Humoral immunity in HIV infection - Introduction

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

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 and preventing infection. 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.

The neutralizing antibody response to HIV-1

Merle Schanz, Thomas Liechti, Michael Huber, Lucia Reh, David Beauparlant, Jacqueline Weber, Therese Uhr, Peter Rusert, Alexandra Trkola

The HIV envelope protein trimer mediates attachment to and entry into target cells and is the only viral protein exposed on the surface of HIV. Neutralizing antibodies directed against the HIV envelope complex can block virus infection, however, elicitation and efficacy of this protecting immune response is impaired in vivo by a number of factors. The envelope trimer adopts a conformation which shields vulnerable domains from antibody access while at the same time the relatively high conformational flexibility of the viral envelope restricts elicitation of relevant B cell responses. However, most importantly, HIV can rapidly mutate and thereby adapt and evade the neutralizing antibody response. While neutralization activity against contemporaneous viral quasi-species is low or not detectable in HIV infected individuals, activity against earlier autologous viruses is frequently detected, highlighting that contemporaneous isolates have escaped antibody pressure. This interplay between virus and host immune response starts immediately after transmission and continues throughout late disease stages. Our work aims to derive a better understanding of how autologous neutralizing antibodies develop during the course of the infection. In particular we are interested to resolve how B cell responses to the newly emerging virus strains escaping the concurrent antibody response are triggered. To this end we study the humoral immune response to the HIV envelope proteins gp120 and gp41 in a cohort of HIV infected individuals who are followed from acute to chronic infection stages. HIV-envelope specific antibody responses are mapped, characterized for binding and autologous and heterologous neutralization activity. Ig genes from HIV envelope specific memory B cells are cloned and lineage evolution and maturation of these antibodies investigated by analyzing the Ig repertoire in these individuals longitudinally by employing next generation sequencing. With these analyses we aim to obtain a detailed insight into the regulation of neutralizing antibody evolution in HIV infection. Information on if and how initial immune responses shape B cell responses to the HIV envelope proteins and what is needed to trigger effective neutralizing antibody responses is urgently needed for HIV vaccine design. Findings from our studies may thus directly impact development of future vaccines.

Most of the neutralizing activity elicited in HIV infection is specific for the autologous virus strains, only few individuals develop potent cross-neutralizing activity, Nevertheless, rare potent monoclonal antibodies with broad cross neutralizing activity have been isolated from infected individuals and are currently considered as the most promising leads towards an effective HIV vaccine. Defining how such broadly cross neutralizing antibodies evolve, their isolation and characterization, definition of their mode of action as well as escape pathways in vivo and in vitro is thus of outmost importance and a focus of this research project.

The role of Fc Receptor mediated antiviral immunity in HIV infection

Thomas Liechti

Cells infected with HIV express envelope trimers on their surface. Opsonization of the viral envelope proteins by antibodies flags infected cells for destruction by immune cells expressing Fc receptors. Antibody-dependent cell-mediated virus inhibition (ADCVI) involves an array of defence mechanisms including antibody dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and the release of antiviral cytokines and chemokines. Although presence of ADCC mediating antibodies in patient sera has been demonstrated for long, to what extent these mechanisms play a role in controlling HIV infection is incompletely understood.

One major obstacle in studying ADCC activity elicited during HIV infection has been the lack of suitable assay systems to quantify ADCC against target cells infected with the autologous virus. Over recent years a number of novel assay systems to measure ADCC have been established, but shortcomings of the assay systems remain, most prominently a low reproducibility due to variation in target and effector cell activity. In the current project we conduct a systematic comparison of ADCC activity during early and late disease stages of HIV infection using a wide spectrum of assay systems which incorporate both measures of FcR stimulation on effector cells as well as target cell destruction to derive a comprehensive picture of ADCC activity in HIV infection. The latter is crucial in order to define whether or not ADCC activating antibody responses need to be elicited by future HIV vaccines.