Alveolar macrophages and the complexity of HCMV populations

A huge number of different human cytomegalovirus strains circulate in humans. Even healthy persons can be infected with more than one HCMV strain remaining in the infected person for the entire life, mostly in a latent state with sporadic reactivation episodes. It is assumed that changes in the HCMV genome may occur over time eventually forming the patients individual HCMV population. HCMV infections are usually asymptomatic in individuals with a healthy immune system but may cause serious illness in patients under severe immunosuppression like patients after lung transplantation. Previous studies have shown that an HCMV infection in lung transplant recipients (LTRs) frequently results from mixed HCMV populations and the mixture may be even more complex when the patient acquires further populations by the donor organ. Infections with mixed HCMV populations seem to enhance clinical symptoms like fever, and leukopenia which finally may lead to HCMV-pneumonia. HCMV populations in individual patients are only limited characterized due to the methods available so far. Hence, the extent of HCMV complexity is far from being understood and it is also not known how complex HCMV populations may influence the pathogenic potential of HCMV. In LTRs, the lung is a very critical site where uncontrolled HCMV infection may start. It is also the site where both, donor- and recipient-derived alveolar macrophages (AMs) reside in the alveolar spaces of the transplanted lung. As recently shown this particular cell type frequently carries HCMV, even in healthy persons. We now hypothesize that HCMV-infected AMs substantially contribute to the complexity of mixed HCMV populations after lung transplantation. The aims of this research proposal are (i) to uncover the HCMV populations which emerge during active HCMV infections by the use of novel and highly sensitive sequencing tools, (ii) to determine the contribution of HCMV-infected AMs therein, and (iii) to assess the growth properties of the respective HCMV strains in cell culture. To this end, multiple left-over samples will be obtained from LTRs after routine surveillance. These clinical samples will be used to characterize HCMV populations present in the bronchoalveolar lavage (BAL) and blood, to investigate HCMV-infected AMs isolated from the BAL, and to determine the growth properties of isolated viruses by a cell culture model. The results gained from this project will improve our understanding of HCMV diversity and how this might influence the pathogenicity of HCMV. This knowledge will form the basis for future studies with regard to HCMV strain-specific immune responses and to the development of novel treatment options for transplant patients.

Alveolar macrophages and the complexity of the HCMV population The human cytomegalovirus (HCMV), a member of the herpesvirus family, consists of numerous genetically different strains. About 50 - 70% of adults in Central Europe develop HCMV-specific antibodies. In HCMV-seropositive people, the virus exists lifelong in a limited number of monocytes without viral replication (latency). The initial infection as well as sporadic virus replications (reactivation) that originate from HCMV-infected monocytes usually are asymptomatic and are controlled by the immune response. Further infections with different HCMV strains (re-infection), however, are not prevented by the specific immunity leading to mixed strain infections. Yet, it is still unclear how many strains are acquired during lifetime and to what extent they differ in their infection behaviour. Organ transplant patients who are under immunosuppressive therapy may suffer from repeated viral replication episodes potentially leading to severe organ damage and chronic organ rejection. Antiviral prophylaxis and therapy is given to stop viral replication but this may cause kidney damage. It is assumed that in lung transplant patients HCMV-infected monocytes that differentiate into alveolar macrophages, play an important role in the virus dissemination. Previous studies reported the transmission of HCMV by the donor organ to HCMV-seronegative recipients, accompanied by a high risk of disease. However, it was largely unknown to what extent HCMV strains from the donor can be transmitted to HCMV-seropositive recipients, whether both sources contribute to virus replication, and how the virus genomes change over time. The overall aim of this project was to determine the strain composition and its replication dynamic after transplantation, to identify the origin of the HCMV strains, and to understand the contribution of the alveolar macrophages. For the first time, we were able to directly show that the donor organ may contain multiple HCMV strains which can be transmitted, regardless of whether the recipient was already HCMV-infected or not. The HCMV population after transplantation can be composed of both, donor- and recipient strains, with all strains replicating similarly well. Also, the genome sequences remain unchanged over time. Remarkably, donor strains can circulate in the blood as early as within the first week after transplantation and recipient's HCMV-infected monocytes and alveolar macrophages colonize the donor organ in the first year after transplantation. Our data show the importance to develop improved strategies to reduce both donor HCMV transmission and reactivation of the recipient strains. While an enormous strain diversity was observed in the entire study population no more than three HCMV strains were present within a single person. This data implies that re-infections with genetically different strains are likely, but that after a few infection cycles there is protection against further re-infections. This findings form the basis for more effective vaccine strategies.