INTRODUCTION

The identification of disease-driving pathogens is even today, after several decades of intensive research, still ongoing and by far not finished. Research on specific agents correlating with common human diseases will need to be enhanced. This applies for e.g. hypervariant forms of viruses – such as specific RNA viruses – generating new or massively modified isotypes with severe and specific pathogenic potential (see e.g. HIV, Hepatitis C and Zika viruses, EBOLA-like viruses with the potential to breach the border between animal and human hosts). Another group of putative risk factors might be of special interest, which may consist of a set of very heterogeneous small episomal DNA molecules which exacerbate their pathogenic function by simultaneous activity of many different representatives of these agents. This could significantly complicate the identification of causative correlations of such agents with disease development, particularly, if the multiplicity of risk molecules is still very low. Only intensive and targeted research can establish such novel etiologies.

We are currently testing a group of highly prevalent molecules called Bovine Meat and Milk Factors  (BMMFs) for a possible link with diseases. BMMFs are frequently isolated from bovine meat and milk samples but also from human material and might represent risk factors for the development of some of the most common human diseases like e.g. breast and colon cancer, but also neurodegenerative disease like Multiple Sclerosis, Alzheimer’s and Parkinson’s Disease. Positive results might result in novel options for diagnosis and specific disease prevention and treatment.

IN THE BEGINNING…

Within several decades of intensive research, Harald zur Hausen and Ethel-Michele de Villiers laid the foundation for the identification and general understanding of a multitude of previously unknown viruses culminating in the 2008 Nobel Prize in Physiology or Medicine for Harald zur Hausen “for his discovery of human papilloma viruses causing cervical cancer” (the other half jointly to Françoise Barré-Sinoussi and Luc Montagnier "for their discovery of human immunodeficiency virus"), paving the way for a new understanding of virology and successful vaccination strategies for cervical cancer (Reference Center and later international WHO reference center for Human Papillomaviruses chaired by E.-M. de Villiers from 1984 to 2013). They further focused on the identification of novel groups of viruses as possible causative agents for common human diseases. A careful analysis of epidemiologic aspects (e.g. dietary habits) of specific human diseases has led to the current focus on isolation and identification of formerly unknown putative infectious agents from bovine meat and milk products and particularly from cancers of the colon and breast. The broad spectrum of identified agents opened up entirely new fields of virology and in 2013, the novel group “Episomal-Persistent DNA in Cancer- and Chronic Diseases” was formed, headed by Harald zur Hausen and Ethel-Michele de Villiers. It identifies novel infectious agents and their potential disease correlations and prevention strategies. In 2018, it is led by Timo Bund with Harald zur Hausen and Ethel-Michele de Villiers as initiators of these activities as consultants.

CURRENT RESEARCH

Isolation of Bovine Meat and Milk Factors

Current Research Isolation of Bovine Meat and Milk Factors

Current Research Isolation of Bovine Meat and Milk Factors
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The group “Episomal Persistent DNA in Cancer- and Chronic Diseases” isolates plasmid-like episomal DNAs mainly from bovine meat and milk but also human sample materials termed Bovine Meat and Milk Factors or shortly BMMFs. Epidemiology correlates the consumption of bovine meat and milk with a higher prevalence of e.g. breast and colon cancer on a global scale, pointing towards a zoonotic infection as possible risk factor for disease development. Interestingly, BMMFs are also isolated from patients suffering Multiple Sclerosis. Additionally, degradation-resistant DNAs comparable to BMMFs were found in scrapie-particle preparations (Manuelidis, J. Neurovirol, 2011) whereas BMMF-related proteins were identified in mouse and hamster CNS as well as human glioblastoma (Yeh et al., PNAS, 2017) indicating a possible link with neuronal diseases.

Functional Characterization of Bovine Meat and Milk Factors

Current Research Functional Characterization of Bovine Meat and Milk Factors

Current Research Functional Characterization of Bovine Meat and Milk Factors
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Basic research projects include the verification of BMMF bioactivity in human cells. Therefore, BMMF replication, transcription and translation experiments are performed based on human cell line models (Eilebrecht at al., Scientific Reports, 2018). Analysis of phenotypic changes in the human host cells are characterized together with specific host transcriptome analysis based on RNA Seq to identify putative key regulators important for general BMMF bioactivity or pathogenic functions. Replication signals, transcription/translation control elements are analyzed together with putative protein-coding open reading frames as basic requirement for protein-protein or protein-DNA/RNA interaction experiments (IP, CHiP, PAR-CLiP, MassSpec). The formation of infective BMMF particles is tested based on particle analysis (Electron Microscopy, Crystallography) and in infection experiments (IF, IHC, FACS, WB).

Establishment of diagnostic tools based on Bovine Meat and Milk Factors

Current Research Establishment of diagnostic tools based on Bovine Meat and Milk Factors

Current Research Establishment of diagnostic tools based on Bovine Meat and Milk Factors
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Selected BMMF genome-encoded proteins serve as markers or targets for establishment of specific serological assays to test human blood. Increased antibody titers against BMMF antigens in the blood samples indicate a general exposure of humans towards BMMF agents and - more specifically - might represent a disease marker with prognostic value for disease outcome enabling novel options for prevention and treatment. In parallel, highly specific antibodies are produced for detection of putative pathogenic BMMF proteins in human (clinical) samples to show a possible BMMF protein function in a broad set of human diseases.

HYPOTHESIS

Plasmid-like episomal Bovine Meat and Milk Factors (BMMF) are taken up within nutrition (dairy or meat products) very early in life and lead to local persistence of BMMFs. While in most cases the immune system is able to clear and avoid high concentrations of such molecules within the tissue, in some individuals a long-lasting presence of BMMFs is plausible. Effectors in this situation might be specific sugars present in human milk (known to provide protection against specific viral and bacterial infections during prolonged breast feeding) or immune-tolerance, which helps certain molecules to evade the immune system to persist in the host. After latency times of several decades and ongoing distribution of BMMFs, induction of local chronic inflammation might be the basis for an increased risk for accumulation of tumorigenic mutations (genomic instabilities) leading to precursor lesions and cancer development. The BMMF-driven occurrence of local infections in specific tissue areas might finally lead to a non-specific and statistical development of mutation-driven tumorigenesis in the affected tissues.

SIGNIFICANT ACCOMPLISHMENTS

Verified BMMF bioactivity in human cells – BMMFs show replication, transcription and protein translation in human cells and induce changes in the host transcriptome.

Detection of BMMF-specific antibodies in human blood indicating exposure of humans towards BMMFs.

Finalized production of a set of highly specific anti-BMMF antibodies ready for high throughput testing of clinical samples for identification of BMMF proteins.

Protein structure of representative BMMF protein solved – new avenues for prevention and inhibitory drug screening.

FUTURE OUTLOOK

The key aspects for identification of specific pathogenic functions of Bovine Meat and Milk Factors (BMMFs) are to understand the mechanism how BMMF infection and persistence takes place. Therefore, infection models will be established to analyze inflammatory response after BMMF infection including specific pathway and receptor analysis. Bringing BMMF research towards translational research and clinical application, we will test for BMMF-correlated predictive scores for specific diseases as basis for preventive and therapeutic strategies like e.g. BMMF-based vaccination or antibody treatment. Strong focus will still be on the ongoing isolation of new BMMFs to complete the list of putative BMMF-related disease markers.

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