Interplay between BK polyomavirus genetic evolution and humoral response: Toward new biomarkers of severe infection and antibody based immunotherapy – BKNAB

BK polyomavirus (BKPyV)-associated nephropathy (PVAN) remains an important cause of kidney transplant (KTx) dysfunction. There are few treatment options available for KTx recipients with sustained high level BKPyV viremia. Clinical management of PVAN relies on reduction of immunosuppressive therapy, with the aim of inducing host antiviral immune response.
Our preliminary results on a prospective cohort suggest that neutralizing antibodies (NAbs) have a protective role against BKPyV infections and that mutations in the VP1 capsid protein are selected during prolonged infection in immunosuppressed patients.
Our hypothesis is that during BKPyV reactivation, expansion of the viral population generates significant intra-patient genetic diversity. An effective immune response may succeed in resolving the viral infection, but alternatively, a limited immune response may allow for the natural selection of viral escape mutants in the VP1 gene, resulting in proteins with modified phenotype and escape from antibody neutralization.
Starting from clinical samples obtained from kidney transplant recipients, our objectives are: i) to show that intra-host BKPyV evolution occurs during prolonged infection; ii) to demonstrate that naturally selected mutations have functional consequences on neutralization and viral entry; and iii) to isolate broadly neutralizing antibodies that can be translated into clinical use to prevent and treat these infections.
We will include adult kidney transplanted patients after a first positive viremia and study the intra-host evolution of BKPyV by deep sequencing of full-length viral genomes on longitudinal urine and plasma samples. Genetic evolution will be compared between patients with clearance of viremia within six months and patients with sustained viremia.
Using pseudotype viruses with VP1 mutations, we will study the cell tropism of different variants, and the ability of patient sera to neutralize these pseudotype viruses. These results will be confirmed with clinical strains from patient’s samples. We will also use structural bioinformatics to investigate the impact of VP1 mutations on capsid structure and receptor binding.
Based on existing data for other viruses, including the closely related JC polyomavirus, we believe that generation of broadly neutralizing monoclonals could have therapeutic potential in the context of BKPyV infection. We propose to produce BKPyV-specific NAbs using fluorescence-labeled VLPs as probes to sort circulating memory B-cells from PBMC of KTx recipients who successfully controlled BKPyV replication. The antibodies expressed by the sorted B-cells will be reconstructed after single cell RT PCR and the neutralizing activity of the expressed human monoclonals will be tested using the pseudotypes produced in WP2. The ability of these antibodies to inhibit virus replication in cell culture will also be tested. Selected NAbs will be patented, with a view to clinical development for the prevention and treatment of BKPyV infections
Our project associates medical and scientific partners with complementary expertise. One of the most innovative aspects of our project is the combination of genetic and functional approaches, as longitudinal analysis of viral quasi-species, coupled with the functional analysis of VP1 variants, has never been performed in the setting of these persistent DNA virus infections. In the field of transplantation medicine, the study aims to resolve two outstanding problems in the clinical management of KTx recipients with BKPyV viremia: how to identify patients at risk for sustained BKPyV replication, and how to treat PVAN after failure of first-line immunotherapy modulation.