Function-guided selection of salivary antigens from Ornithodoros erraticus argasid ticks and assessment of their protective efficacy in rabbits.

The identification of new protective antigens for the development of tick vaccines may be approached by selecting antigen candidates that have key biological functions. Bioactive proteins playing key functions for tick feeding and pathogen transmission are secreted into the host via tick saliva. Adult argasid ticks must resynthesise and replace these proteins after each feeding to be able to repeat new trophogonic cycles. Therefore, these proteins are considered interesting antigen targets for tick vaccine development. In this study, the salivary gland transcriptome and saliva proteome of Ornithodoros erraticus females were inspected to select and test new vaccine candidate antigens. For this, we focused on transcripts overexpressed after feeding that encoded secretory proteins predicted to be immunogenic and annotated with functions related to blood ingestion and modulation of the host defensive response. Completeness of the transcript sequence, as well as a high expression level and a high fold-change after feeding were also scored resulting in the selection of four candidates, an acid tail salivary protein (OeATSP), a multiple coagulation factor deficiency protein 2 homolog (OeMCFD2), a Cu/Zn-superoxide dismutase (OeSOD) and a sulfotransferase (OeSULT), which were later produced as recombinant proteins. Vaccination of rabbits with each individual recombinant antigen induced strong humoral responses that reduced blood feeding and female reproduction, providing, respectively, 46.8%, 45.7%, 54.3% and 31.9% protection against O. erraticus infestations and 0.7%, 3.9%, 3.1% and 8.7% cross-protection against infestations by the African tick, Ornithodoros moubata. The joint protective efficacy of these antigens was tested in a second vaccine trial reaching 58.3% protection against O. erraticus and 18.6% cross-protection against O. moubata. These results (i) provide four new protective salivary antigens from argasid ticks that might be included in multi-antigenic vaccines designed for the control of multiple tick species; (ii) reveal four functional protein families never tested before as a source of protective antigens in ticks; and (iii) show that multi-antigenic vaccines increase vaccine efficacy compared with individual antigens. Finally, our data add value to the salivary glands as a protective antigen source in argasids for the control of tick infestations.

A proteomics informed by transcriptomics insight into the proteome of Ornithodoros erraticus adult tick saliva.

BACKGROUND: The argasid tick Ornithodoros erraticus is the main vector of tick-borne human relapsing fever (TBRF) and African swine fever (ASF) in the Mediterranean Basin. The prevention and control of these diseases would greatly benefit from the elimination of O. erraticus populations, and anti-tick vaccines are envisaged as an effective and sustainable alternative to chemical acaricide usage for tick control. Ornithodoros erraticus saliva contains bioactive proteins that play essential functions in tick feeding and host defence modulation, which may contribute to host infection by tick-borne pathogens. Hence, these proteins could be candidate antigen targets for the development of vaccines aimed at the control and prevention of O. erraticus infestations and the diseases this tick transmits. The objective of the present work was to obtain and characterise the proteome of the saliva of O. erraticus adult ticks as a means to identify and select novel salivary antigen targets. METHODS: A proteomics informed by transcriptomics (PIT) approach was applied to analyse samples of female and male saliva separately using the previously obtained O. erraticus sialotranscriptome as a reference database and two different mass spectrometry techniques, namely liquid chromatography-tandem mass spectrometry (LC-MS/MS) in data-dependent acquisition mode and sequential window acquisition of all theoretical fragment ion spectra MS (SWATH-MS). RESULTS: Up to 264 and 263 proteins were identified by LC-MS/MS in the saliva of O. erraticus female and male ticks, respectively, totalling 387 non-redundant proteins. Of these, 224 were further quantified by SWATH-MS in the saliva of both male and female ticks. Quantified proteins were classified into 23 functional categories and their abundance compared between sexes. Heme/iron-binding proteins, protease inhibitors, proteases, lipocalins and immune-related proteins were the categories most abundantly expressed in females, while glycolytic enzymes, protease inhibitors and lipocalins were the most abundantly expressed in males. Ninety-seven proteins were differentially expressed between the sexes, of which 37 and 60 were overexpressed in females and males, respectively. CONCLUSIONS: The PIT approach demonstrated its usefulness for proteomics studies of O. erraticus, a non-model organism without genomic sequences available, allowing the publication of the first comprehensive proteome of the saliva of O. erraticus reported to date. These findings confirm important quantitative differences between sexes in the O. erraticus saliva proteome, unveil novel salivary proteins and functions at the tick-host feeding interface and improve our understanding of the physiology of feeding in O. erraticus ticks. The integration of O. erraticus sialoproteomic and sialotranscriptomic data will drive a more rational selection of salivary candidates as antigen targets for the development of vaccines aimed at the control of O. erraticus infestations and the diseases it transmits.

Proteomics informed by transcriptomics for a qualitative and quantitative analysis of the sialoproteome of adult Ornithodoros moubata ticks.

BACKGROUND: The argasid tick Ornithodoros moubata is the main vector in mainland Africa of African swine fever virus and the spirochete Borrelia duttoni, which causes human relapsing fever. The elimination of populations of O. moubata would contribute to the prevention and control of these two serious diseases. Anti-tick vaccines are an eco-friendly and sustainable means of eliminating tick populations. Tick saliva forms part of the tick-host interface, and knowledge of its composition is key to the identification and selection of vaccine candidate antigens. The aim of the present work is to increase the body of data on the composition of the saliva proteome of adult O. moubata ticks, particularly of females, since in-depth knowledge of the O. moubata sialome will allow the identification and selection of novel salivary antigens as targets for tick vaccines. METHODS: We analysed samples of female and male saliva using two different mass spectrometry (MS) approaches: data-dependent acquisition liquid chromatography-tandem MS (LC-MS/MS) and sequential window acquisition of all theoretical fragment ion spectra-MS (SWATH-MS). To maximise the number of proteins identified, a proteomics informed by transcriptomics analysis was applied using the O. moubata salivary transcriptomic dataset previously obtained by RNA-Seq. RESULTS: SWATH-MS proved to be superior to LC-MS/MS for the study of female saliva, since it identified 61.2% more proteins than the latter, the reproducibility of results was enhanced with its use, and it provided a quantitative picture of salivary components. In total, we identified 299 non-redundant proteins in the saliva of O. moubata, and quantified the expression of 165 of these in both male and female saliva, among which 13 were significantly overexpressed in females and 40 in males. These results indicate important quantitative differences in the saliva proteome between the sexes. CONCLUSIONS: This work expands our knowledge of the O. moubata sialome, particularly that of females, by increasing the number of identified novel salivary proteins, which have different functions at the tick-host feeding interface. This new knowledge taken together with information on the O. moubata sialotranscriptome will allow a more rational selection of salivary candidates as antigen targets for tick vaccine development.

RNA-seq analysis and gene expression dynamics in the salivary glands of the argasid tick Ornithodoros erraticus along the trophogonic cycle.

BACKGROUND: The argasid tick Ornithodoros erraticus is the main vector of tick-borne human relapsing fever (TBRF) and African swine fever (ASF) in the Mediterranean Basin. Tick salivary proteins secreted to the host at the feeding interface play critical roles for tick feeding and may contribute to host infection by tick-borne pathogens; accordingly, these proteins represent interesting antigen targets for the development of vaccines aimed at the control and prevention of tick infestations and tick-borne diseases. METHODS: To identify these proteins, the transcriptome of the salivary glands of O. erraticus was de novo assembled and the salivary gene expression dynamics assessed throughout the trophogonic cycle using Illumina sequencing. The genes differentially upregulated after feeding were selected and discussed as potential antigen candidates for tick vaccines. RESULTS: Transcriptome assembly resulted in 22,007 transcripts and 18,961 annotated transcripts, which represent 86.15% of annotation success. Most salivary gene expression took place during the first 7 days after feeding (2088 upregulated transcripts), while only a few genes (122 upregulated transcripts) were differentially expressed from day 7 post-feeding onwards. The protein families more abundantly overrepresented after feeding were lipocalins, acid and basic tail proteins, proteases (particularly metalloproteases), protease inhibitors, secreted phospholipases A2, 5'-nucleotidases/apyrases and heme-binding vitellogenin-like proteins. All of them are functionally related to blood ingestion and regulation of host defensive responses, so they can be interesting candidate protective antigens for vaccines. CONCLUSIONS: The O. erraticus sialotranscriptome contains thousands of protein coding sequences-many of them belonging to large conserved multigene protein families-and shows a complexity and functional redundancy similar to those observed in the sialomes of other argasid and ixodid tick species. This high functional redundancy emphasises the need for developing multiantigenic tick vaccines to reach full protection. This research provides a set of promising candidate antigens for the development of vaccines for the control of O. erraticus infestations and prevention of tick-borne diseases of public and veterinary health relevance, such as TBRF and ASF. Additionally, this transcriptome constitutes a valuable reference database for proteomics studies of the saliva and salivary glands of O. erraticus.