[Protection against Lyme borreliosis – analysis of the most important pathogen vector in Europe, the tick Ixodes ricinus, and identification of vaccine candidates]. (Doctoral thesis)
- Clinical and Applied Virology
Ixodes ricinus is the most important pathogen vector in Europe and transmits a wide range of pathogenic bacteria, viruses and protozoa. The diseases caused by these pathogens can have serious consequences for human health, but they also threaten productivity and/or welfare of livestock, wildlife and companion animals. With around 65,500 diagnosed patients annually in Europe, and a high number of unreported cases, Lyme borreliosis is the most prevalent vector-borne disease in the Northern hemisphere. The causative agent, the bacteria Borrelia burgdorferi sensu lato, are transmitted to humans and animals via the tick I. ricinus. In addition, tick-borne encephalitis, babesiosis, rickettsiosis, human granulocytic anaplasmosis and tularemia are caused by pathogens transmitted by I. ricinus.
The prevalence of ticks is rising in Europe. Concurrently, intensively used tick repellents lose their efficacy due to the emergence of resistant tick populations. The development of new acaricides is highly intense in costs and thus stagnated. Moreover, acaricide residues were found in milk and meat products from treated animals besides contaminating the environment. Since acaricides are getting more and more limited in efficacy, contaminate the environment and do not protect against the transmission of diseases, new strategies are needed to control ticks and as a consequence tick-borne diseases. Here, anti-tick vaccines could be an effective and environmentally friendly alternative. However, the development of such a vaccine was aggravated and so far prevented by the lack of information about the tick genome, transcriptome and proteome.
In the present study, the first reference genome of this important tick species was obtained by performing whole genome sequencing in a shotgun and de novo assembly approach. In a second genome sequencing and analysis approach, the genome contigs were combined into scaffolds with ultra-long PacBio reads from 3rd generation sequencing. Thereby, the total genome length spanned was increased by more than 30 %. This resulted in an increased biological value of the genome draft as blast searches and alignment approaches are facilitated. In addition, more than 6,000 putative genes were predicted in I. ricinus by transcript discovery and more than 25,000 potential genes were annotated based on homology analysis to the related species Ixodes scapularis, thus representing the first genome annotation approach in I. ricinus.
The genome sequences were further complemented with genome size estimation by flow cytometry of laboratory reared and field collected ticks. This analysis revealed the genome of I. ricinus to be with 2.65 Gb around 500 Mb larger than the genome of its American relative I. scapularis, despite the same number of chromosomes. Since there was a high homology in coding regions observed between I. ricinus and I. scapularis, this difference in genome size by 26 % most likely results from multiplication events in non-coding genome regions after separation of the species. Laboratory rearing or geographical origin had no measurable impact on the I. ricinus genome size. In contrast, the genome of the I. ricinus cell line IRE/CTVM19 was shown to be 1.4 fold the size of the I. ricinus tick genome. It is possible that multiplication of repeats or insertion of transposable elements across the genome are more efficient in a rapidly replicating cell in culture than during natural evolution.
In addition to the reference genome, a reference database for the identification of antibody inducing midgut proteins present at an early feeding stage, i.e. in the naïve midgut, was created in this study. In this first large scale annotation and system analysis approach of the unfed I. ricinus midgut, more than 10,000 transcripts and 285 proteins were annotated for their molecular function, cellular localization and/or biological process they are involved in.
Subsequently, the databases were applied for the identification of vaccine candidates against this important pathogen vector. Vaccine candidates against I. ricinus were identified in vivo by immunization of mice with tick midgut membrane extract followed by challenge experiments. A reduction in feeding success could be observed. Thus, serological antibody targets were identified by mass spectrometry using the established databases as reference. Additionally, an in silico reverse vaccinology approach was designed to first screen the databases for sequences coding for proteins located at the membrane. Then, surface exposed (in order to be accessible to antibodies entering the midgut with the blood) and antigenic peptides of these proteins, which are abundant in epitopes, were selected. Candidates were investigated for their homology to human and mouse in order to prevent autoimmunogenic effects. The sequences of 5 candidates identified in vivo and 5 candidates identified in silico were synthetized and incorporated in a vaccine scaffold consisting of Hepatitis B virus (HBV) tandem core proteins. These shell core proteins carry the vaccine candidates on their surface and form highly immunogenic virus-like particles (VLPs). Since the identified candidates are membrane proteins, expression, correct folding and presentation to the immune system are not trivial. The specific design of the HBV tandem core technology allows the insertion and exposition of correctly folded whole proteins and hydrophobic peptides whilst proper assembly of VLPs in E. coli. By using the HBV-VLP technology, the vaccine candidates are moreover inserted into a vector with high immunogenic properties that are transferred to the antigens carried.
In summary, besides the establishment of comprehensive databases for genome, transcriptome and proteome and the first genome size estimation in I. ricinus, this dissertation advances the development of an anti-I. ricinus vaccine by the identification of promising vaccine candidates. By their insertion into a suitable vaccine scaffold, the way for further immunization studies and pre-clinical analysis of the identified promising vaccine candidates was paved.