Glycoprotein incorporation into retroviral particles: mechanisms and applications
Our group has a long-standing interest in viral glycoprotein (Env) function and biogenesis and in the processes involved in glycoprotein incorporation into viral particles (in particular into HIV-1 particles). At present we focus on two main projects:
1. "Preclinical evaluation of a vaccine strategy based on conserved conformational epitopes of the HIV envelope glycoprotein"
HIV Env is the target antigen against which neutralizing antibodies are directed. Numerous strategies employing monomeric or trimeric Env proteins or native inactivated virions have failed to induce broadly neutralizing responses. This is largely due to the immense and rapidly developing sequence diversity of HIV in vivo, so that induced immune responses, effective against the HIV strain from which the Env vaccine has been derived, generally do not prevent infection by another HIV strain. The conceptual basis of the vaccine strategy followed here is that one or several Env structures, which exclusively arise during the dynamic interaction process of Env with the cellular HIV receptor complex (Fig.1), may represent potent conformationally conserved immunogens for the induction of broadly cross-neutralizing antibodies. Non-infectious HIV pseudovirions (PV) have been chosen as the basis for preparing vaccine preparations enriched in induced Env epitopes. In PV, all of the required protein components (Env, CD4, coreceptor) are embedded in a lipid membrane, a fact which may be a prerequisite for the induction of relevant Env epitopes. Importantly, we have previously demonstrated that membrane fusion can occur between PV which have incorporated HIV-Env and human CD4/coreceptor, respectively. Thus in the approach employed here, non-infectious HIV PV containing fusion-defective mutant Env (Env*) are exposed to non-infectious HIV PV containing the HIV receptor complex (human CD4/CCR5 or human CD4/CXCR4). The use of defective mutant Env ensures that the cascade of conformational changes in Env, which are induced on binding to the cellular receptor complex, be terminated at an intermediate stage (Fig.2). In order to prepare the vaccine, Env*-PV are mixed and incubated with CD4/coreceptor-PV. During this time, new Env epitopes are induced and, depending on the mutant Env* employed, are “fixed” at a defined intermediate conformation. We have confirmed that induced Env epitopes are actually present in the PV vaccine preparation by analysing the binding of specific antibodies (e.g. CG10) to the PV vaccine and, as negative control, untreated PV preparations (Fig. 3).
The PV vaccine contains human CD4 and coreceptor molecules and antibodies elicited against these proteins interfere with Env-specific virus neutralisation assays. Human CD4/coreceptor-transgenic rats (developed by our collaboration partner Dr. O.T. Keppler, University of Heidelberg) are tolerant to these proteins so that it is possible to analyse neutralising serum IgG responses after immunisation of these animals with the PV vaccine. Additionally, since these small animals support HIV replication in vivo, albeit to low levels, it is possible to easily evaluate the response of vaccinated animals to HIV challenge.
The aims of this project at present are 1. to establish, first in non-transgenic mice, the adjuvant formulations and routes of immunisation which yield maximal antibody responses (in collaboration with Dr. C. A, Guzman, GBF-German Research Centre for Biotechnology, Braunschweig). 2. to test promising formulations in the transgenic rat model for their ability to induce antibodies capable of neutralising homologous and, in particular, heterologous HIV strains. 3. to develop further vaccines with alternative Env mutants presumably presenting different intermediate fusion conformations and test these as above and 4. in a collaborative effort, to combine the most promising pseudovirion vaccine candidates with other HIV vaccine components (Tat, Nef) which alternatively aim at reducing HIV viral load and delaying disease progression.
2. “Role of the HIV Env C-terminus in infection and replication”
In contrast to most other enveloped viruses, the surface glycoproteins of lentiviruses, including human immunodeficiency virus (HIV), contain very long C-terminal cytoplasmic tails (CTs) of about 100-200 aa, which are absolutely required for virus replication in vivo (Fig. 4). Additionally, in most T-cell lines in vitro (these cells are referred to as non-permissive cells), mutant HIV, encoding C-terminally truncated Env, cannot establish a spreading infection. However, in some few T-cell lines spreading infection can occur (these cells are referred to as permissive cells). Thus the general aim of this project is to gain insight as to the essential processes mediated by the Env-CT and to understand the basis of the permissivity/non-permissivity phenotype of different T-cell lines.
Our ongoing analysis of the properties of mutant HIVs encoding Env proteins truncated within the CT indicate that viral spread by cell-cell transmission may be more strongly impacted by the HIV-Env CT truncation than the infectivity of cell-free virions. Thus in one specific aspect of this project, different methodologies will be employed to analyse cell-cell transmission. These will include virological assays, confocal microscopic assays and procedures to alter and to analyse signal transduction pathways which may play a role in viral transmission.
In another part of the project, screening procedures to identify putative cellular interaction partners of the Env-CT will be implemented. Tagged and untagged versions of the Env-CT protein expressed in non-permissive cells, should be enriched, together with associated cellular proteins, by affinity purification from mild detergent lysates of cells. Candidate interaction partners, if observed, should identified by mass spectrometry. Their validity as interaction partners and their possible requirement for Env-CT function will be confirmed using standard procedures.