Molecular Virology – approaches for new diagnostic tools and towards new targeted drugs
We focus on aspects of the HIV life cycle that are critical for replication and its response to antiretroviral drugs.
1. Reliable tropism testing for the clinics – This major effort bases on the fact that HIV occurs as two distinct forms differing in their receptor dependence. The new HIV inhibitor class targets only CCR5-tropic viruses. This necessitates prior to therapy a tropism determination: Does the virus use CCR5 or the alternative CXCR4 chemokine receptor. We set out to establish a unique, reliable diagnostic test together with our spinoff InPheno AG. The tropism-determining sequence “V3” within the HIV envelope gene is rep- resented in a labeled probe, which is hybridized to a patient-derived blood sample. Our system reads CCR5-/CXCR4-tropism and identifies/dissects mixed virus populations, a main advantage over sequencing systems (Fig1). In 2011 our test passed a European ring trial for quality control; it became accredited and accepted by the Swiss BAG for reimbursement through the Analysenliste.
2. Residual virus despite successful therapy? – In some patients a full suppression of HIV is not achieved although medication appears appropriate. In vitro observations suggest that viral expression from integrated genomes could contribute to this worrisome residual viral RNA, which must not necessarily be a sign of treatment failure. Retroviruses such as HIV require as essential step the genomic integration into the host chromosome. The new integrase inhibitors block this key step possibly leading to the first step to- wards HIV genome elimination and eradication. However, in a new project we have identified evidence that even unintegrated viral DNA can allow viral gene expression and potentially also the generation of infectious progeny. Our current investigations attempt to verify production of infectious particles despite the presence of therapeutic doses of integrase inhibitors. We will further try to isolate infectious virus from patients with persisting low viremia. This would have important implications for the clinics.
3. Role of HIV Gag in therapy resistance – It is being recognized that even in times of a broad therapy coverage with the highly potent drug class of HIV protease inhibitors (PI) a fair number of patients fail virologically in the absence of signature mutations in the viral protease gene. For addressing this issue we initiated a collaborative project with the Swiss HIV Cohort Study, in which we systematically analyzed virus sequences near the 3’ end of the gag gene, where key cleavage sites are encode. The anonymized HIV-1 databases of 2000 sequences stems from a characterized therapy context or from untreated patients. Side-by-side analysis of these two groups enables us to identify alterations emerging only under drug pressure. Mutations can enhance “minor protease mutations” or are by themselves responsible for drug-resistance and clinical therapy failure. Our newly identified mutations associate with clinical PI resistance; they either correspond with earlier publi- cations thus validating our algorithm or they have not been described before as treatment-associated escape mutations (Fig2). Currently we are introducing these mutations into lab strains of HIV-1 in order to phenotypically test if they convey PI resistances. We will further isolate infectious virus from patients with persisting low viremia. This could help understanding “unexplained virological therapy failures” and would be an important addition to the current resistance algorithms.
4. A somewhat “exotic” project aims at widening our views on drug discovery: In a collaboration with the South African CSIR we focus on respi- ratory infections caused by RSV and influenza. Together with InPheno we have now been able to validate a new phenotypic screening system for inhibitors. Various indigenous plants, claimed to possess anecdotal activity, have been extracted and tested in a first pilot study. First promising hits have been identified (Fig3) with selective antiviral activity in the absence of toxicity in cell models and await now further profiling and purification. As long-term goal we aim at a pharmaceutical development.
Diagnostic Tools for improved HIV disease management; new concepts for respiratory diseases
My research group has in the past years developed sever- al key methodologies for analysing HIV therapy. In particular the development of tests for viral drug resistance and for identifying the viruses cell tropism are assisting clinicians for disease management. Emerging from this clinical utility we have focussed research onto newer underlying mechanisms of persisting low viral replication in long-term treated patients or on the predictive value of analyses of viral parameters in cells rather than cell-free blood plasma. Also pilot studies on longer term effects of integrated versus unintegrated viral genomes in the patient may help to understand viral progres- sion or may be useful for concepts towards HIV elimination.
Beyond HIV as human pathogen part of our research can directly be applied to other viruses with clinical importance. In fruitful international collaborations we have invested in the identification of new inhibitory concepts targeting vital virus functions. Herein we concentrate on respiratory pathogenic viruses such as Influenza, parainfluenza, and RSV. With a group in Pretoria we recognize the intriguing source of natural compounds in South African plants, known in traditional medicine to possess disease-fighting properties. Our plan is to identify therein the active principles and make them avail- able for pharmaceutical exploitation.