Self-assembled protein arrays from an Ornithodoros moubata salivary gland expression library.

Protein interactions play a critical role in the regulation of many biological events and their study in a high-throughput format has become a key area of proteomic research. Nucleid Acid Programmable Protein Arrays (NAPPA) technology allows the construction of protein arrays from cDNA expression libraries in high-throughput cell-free systems to study protein interaction and functions. Tick saliva contains antihemostatic, anti-inflammatory, and immunosuppressive proteins that counteract the host hemostatic, immune, and inflammatory responses allowing the ingestion of host blood and facilitating its infection by the tick-borne pathogens. Identification of such proteins and their functions could help in the selection of antigenic targets for the development of antitick and transmission-blocking vaccines. With that aim, we have prepared a cDNA expression library from the salivary glands of Ornithodoros moubata and subsequently produced a self-assembled protein microarray using 480 randomly selected clones from that library. The reproducibility of the array, its representativeness of the tick salivary protein repertoire, and the functionality of the in situ expressed proteins have been checked, demonstrating that it is a suitable tool for the identification and functional characterization of soft tick salivary molecules that interact with host proteins. Several clones in the array were shown to bind to human recombinant P-selectin. One of them was a likely secreted tick phospholipase A2, which may represent a potential new ligand for P-selectin. As these salivary molecules are likely involved in blood meal acquisition through the modulation of the host immune and hemostatic responses, this new high-throughput tool could open new avenues for development of new therapeutic agents and control strategies against ticks and tick-borne pathogens.

New salivary anti-haemostatics containing protective epitopes from Ornithodoros moubata ticks: Assessment of their individual and combined vaccine efficacy.

Ornithodoros moubata is the main vector of the pathogens causing African swine fever and human relapsing fever in Africa. The development of an efficient vaccine against this tick would facilitate its control and the prevention of the diseases it transmits to a considerable extent. Previous efforts to identify vaccine target candidates led us to the discovery of novel salivary proteins that probably act as anti-haemostatics at the host-tick interface, including a secreted phospholipase A2 (PLA2), a 7DB-like protein (7DB-like), a riboprotein 60S L10 (RP-60S), an apyrase (APY), and a new platelet aggregation inhibitor peptide, designated mougrin (MOU). In this work, the corresponding recombinant proteins were expressed in Escherichia coli and their individual vaccine efficacy was tested in rabbit vaccination trials. All of them, except the less immunogenic RP-60S, induced strong humoral responses that reduced tick feeding and survival, providing vaccine efficacies of 44.2%, 43.2% and 27.2%, 19.9% and 17.3% for PLA2, APY, MOU, RP-60S and 7DB-like, respectively. In the case of the more protective recombinant antigens (PLA2, APY and MOU), the immunodominant protective linear B-cell epitopes were identified and their combined vaccine efficacy was tested in a second vaccine trial using different adjuvants. In comparison with the best efficacy of individual antigens, the multicomponent vaccine increased vaccine efficacy by 13.6%, indicating additive protective effects rather than a synergistic effect. Tick saliva inoculated during natural tick-host contacts had a boosting effect on vaccinated animals, increasing specific antibody levels and protection.

Identification of protective linear B-cell epitopes on the subolesin/akirin orthologues of Ornithodoros spp. soft ticks.

Subolesin/akirin is a protective antigen that is highly conserved across hematophagous vector species and is therefore potentially useful for the development of a universal vaccine for vector control, including soft ticks. Recent results have shown that in Ornithodoros erraticus and O. moubata soft ticks, RNAi-mediated subolesin gene knockdown inhibits tick oviposition and fertility by more than 90%; however, vaccination with recombinant subolesins resulted in remarkably low protective efficacies (5-24.5% reduction in oviposition). Here we report that vaccination with subolesin recombinants induces non-protective antibodies mainly directed against immunodominant linear B-cell epitopes located on highly structured regions of the subolesin protein, probably unrelated to its biological activity, while leaving the unstructured/disordered regions unrecognized. Accordingly, for a new vaccine trial we designed four synthetic peptides (OE1, OE2, OM1 and OM2) from the unrecognized/disordered regions of the Ornithodoros subolesin sequences and coupled them to keyhole limpet haemocyanin (KLH). These KLH-peptide conjugates induced the synthesis of antibodies that recognized linear B-cell epitopes located on the unstructured loops of the subolesin protein and provided up to 70.1% and 83.1% vaccine efficacies in O. erraticus and O. moubata, respectively. These results show that the protective effect of subolesin-based vaccines is highly dependent on the particular epitope recognized by antibodies on the subolesin sequence and strongly suggest that the biological activity of subolesin is exerted through its unstructured regions. The results reported here contribute to our understanding of the mechanism of protection of subolesin-based vaccines and reveal novel protective peptides that could be included among the array of candidate antigens useful for developing anti-vector vaccines based on subolesin/akirin.

Development of vaccines against Ornithodoros soft ticks: An update.

Ticks are parasites of great medical and veterinary importance since they are vectors of numerous pathogens that affect humans, livestock and pets. Among the argasids, several species of the genus Ornithodoros transmit serious diseases such as tick-borne human relapsing fever (TBRF) and African Swine Fever (ASF). In particular, Ornithodoros erraticus is the main vector of these two diseases in the Mediterranean while O. moubata is the main vector in Africa. The presence of these Ornithodoros ticks in domestic and peridomestic environments may greatly hinder the eradication of TBRF and ASF from endemic areas. In addition, there is a constant threat of reintroduction and spreading of ASF into countries from where it has been eradicated (Spain and Portugal) or where it was never present (the Caucasus, Russia and Eastern Europe). In these countries, the presence of Ornithodoros vectors could have a tremendous impact on ASF transmission and long-term maintenance. Therefore, elimination of these ticks from at least synanthropic environments would contribute heavily to the prevention and control of the diseases they transmit. Tick control is a difficult task and although several methods for such control have been used, none of them has been fully effective against all ticks and the problems they cause. Nevertheless, immunological control using anti-tick vaccines offers an attractive alternative to the traditional use of acaricides. The aim of the present paper is to offer a brief overview of the current status in control measure development for Ornithodoros soft ticks, paying special attention to the development of vaccines against O. erraticus and O. moubata. Thus, our contribution includes an analysis of the chief attributes that the ideal antigens for an anti-tick vaccine should have, an exhaustive compilation and analysis of the scant anti-soft tick vaccine trials carried out to date using both concealed and salivary antigens and, finally, a brief description of the new reverse vaccinology approaches currently used to identify new and more effective protective tick antigens.

Cloning and characterization of a plasminogen-binding enolase from the saliva of the argasid tick Ornithodoros moubata.

Significant amounts of enolase have recently been found in the saliva of the argasid tick Ornithodoros moubata, raising the question as to what the function of enolase in the tick-host interface is. Enolase is a multifunctional glycolytic enzyme known to act as a plasminogen receptor on cellular surfaces, promoting fibrinolysis and extracellular matrix degradation. Fibrinolysis could be important for ticks to dissolve clots that might be formed during feeding as well as to prevent clotting of the ingested blood meal in the tick midgut. Additionally, enolase-mediated extracellular matrix degradation could contribute to the tick feeding lesion. Moreover, previous observations suggested an additional antihaemostatic role for O. moubata enolase as a P-selectin antagonist ligand. Accordingly, the aim of the present study was to investigate the potential role of the O. moubata salivary enolase as a plasminogen receptor and P-selectin ligand, and to evaluate its potential as an antigen target for anti-O. moubata vaccines. The study included the cloning, sequencing and recombinant production of the O. moubata enolase, plasminogen binding and activation assays, P-selectin binding assays, animal immunization trials, and RNAi knockdown of the enolase gene. Here we confirmed that enolase is secreted to the saliva of the tick and provide convincing evidence for a role of this salivary enolase as a plasminogen receptor, most likely stimulating host fibrinolysis and maintaining blood fluidity during tick feeding. The RNAi experiments and immunization trials indicated that enolase could be also involved in the regulation of tick reproduction, suggesting new potential control strategies. Finally, the P-selectin binding experiments demonstrated that this enolase is not a P-selectin ligand.

Protein arrays as tool for studies at the host-pathogen interface.

Pathogens and parasites encode a wide spectrum of multifunctional proteins interacting to and modifying proteins in host cells. However, the current lack of a reliable method to unveil the protein-protein interactions (PPI) at the host-pathogen interface is retarding our understanding of many important pathogenic processes. Thus, the identification of proteins involved in host-pathogen interactions is important for the elucidation of virulence determinants, mechanisms of infection, host susceptibility and/or disease resistance. In this sense, proteomic technologies have experienced major improvements in recent years and protein arrays are a powerful and modern method for studying PPI in a high-throughput format. This review focuses on these techniques analyzing the state-of-the-art of proteomic technologies and their possibilities to diagnose and explore host-pathogen interactions. Major technical advancements, applications and protocol concerns are presented, so readers can appreciate the immense progress achieved and the current technical options available for studying the host-pathogen interface. Finally, future uses of this kind of array-based proteomic tools in the fight against infectious and parasitic diseases are discussed.

An insight into the proteome of the saliva of the argasid tick Ornithodoros moubata reveals important differences in saliva protein composition between the sexes.

Tick saliva contains pharmacologically active molecules that allow these parasites to obtain a blood meal from the host and facilitate host infection by tick-borne pathogens. Recent transcriptomic and proteomic analyses of the salivary glands of several tick species have provided data sets that are invaluable for a better understanding of tick sialomes and tick-host-pathogen relationships. Here we performed a proteomic study of the saliva from the argasid tick Ornithodoros moubata. Saliva samples from female and male specimens were analyzed separately by LC-MS/MS before and after their equalization to facilitate the identification of the less abundant proteins. We report the array of 193 proteins identified in the saliva of O. moubata showing: (i) the broad and complex composition of the saliva of this tick, in good agreement with the complexity of the argasid and ixodid sialomes described previously; (ii) a notable difference in the saliva proteomes of females and males, since only 10 of the proteins identified appeared to be shared by both sexes; and (iii) the presence in the salivary fluid of a wide range of proteins known to be housekeeping/intracellular, which could be secreted in unconventional ways, including exosome secretion.

Subolesin/akirin orthologs from Ornithodoros spp. soft ticks: cloning, RNAi gene silencing and protective effect of the recombinant proteins.

Subolesin/akirin is a well characterized protective antigen highly conserved across vector species and thus potentially useful for the development of a broad-spectrum vaccine for the control of arthropod infestations including hard ticks, mosquitoes, sand flies and the poultry red mite Dermanyssus gallinae. Soft ticks could be also targeted by this vaccine if proved that the soft tick subolesin orthologs are conserved and induce protective immune responses too. However, to date no soft tick subolesin orthologs have been fully characterized nor tested as recombinant antigens in vaccination trials. The objectives of the present work were to clone and characterize the subolesin orthologs from two important vector species of soft ticks as Ornithodoros erraticus and O. moubata, to evaluate the effect of subolesin gene silencing by RNAi, and to test the protective value of the recombinant antigens in vaccination trials. The obtained results demonstrate that both soft tick subolesins are highly conserved showing more than 69% and 74% identity with those of hard ticks in their nucleotide and amino acid sequences, respectively. Additionally, we demonstrate that both soft ticks possess fully operative RNAi machinery, and that subolesin gene silencing by dsRNA injection inhibits oviposition indicating the involvement of subolesin in tick reproduction. Finally, vaccination with the recombinant soft tick subolesins induced a partial protective effect resulting in the reduction of the oviposition rate. These preliminary results encourage further studies on the use of recombinant subolesins as vaccines for the control of soft tick infestations, either alone or in combination with other specific molecules.

Cloning, characterization and diagnostic performance of the salivary lipocalin protein TSGP1 from Ornithodoros moubata.

The argasid tick Ornithodoros moubata is distributed throughout South and East Africa and Madagascar, where it colonizes wild and domestic habitats and feeds on warthogs, domestic swine, and humans. This argasid transmits the spirochete Borrelia duttonii, causing East African tick-borne relapsing fever in humans, and the African swine fever virus, which causes a highly lethal haemorrhagic disease in pigs. Tick surveillance and the elimination of O. moubata from synanthropic environments (human dwellings and pigsties) would facilitate the control and prevention of these two diseases. Since direct surveillance methods are impractical in this context, the development of an indirect method for the detection of specific antibodies against tick salivary proteins in samples taken from animal or human hosts living in the area under study would provide a more convenient surveillance and diagnostic tool. Previous work has indicated that the 20A1 salivary antigen of O. moubata could be an optimal candidate for the development of a specific serological test and identified it as an orthologue of the Ornithodoros savignyi TSGP1 lipocalin. The objectives of the present work were to clone, sequence, and molecularly characterize the O. moubata TSGP1, as well as its production as a recombinant protein in order to assess its usefulness as a diagnostic antigen in an ELISA test for tick surveillance. Our results show that O. moubata TSGP1 (OmTSGP1) conserves the tertiary structure of lipocalins and contains the biogenic amine-binding motif. We also show that OmTSGP1 shares 65% sequence identity with the O. savignyi TSGP1, demonstrating that they represent orthologous proteins and suggesting they share identical function as biogenic amine scavengers. A recombinant form of OmTSGP1 was produced, showing 100% sensitivity and 99.4% specificity in an ELISA test for the detection of anti-O. moubata antibodies in pig sera. This recombinant antigen represents a promising epidemiological tool for O. moubata surveillance that may help to implement control measures against O. moubata-borne diseases.