Targeting the tick protective antigen subolesin reduces vector infestations and pathogen infection by Anaplasma marginale and Babesia bigemina

The ultimate goal of vector vaccines is the control of vector infestations while reducing pathogen infection and transmission to protect against the many diseases caused by vector-borne pathogens. Previously (Vaccine 2011;29:2248-2254), we demonstrated that subolesin vaccination and release of tick larvae after subolesin knockdown by RNA interference (RNAi) were effective for the control of cattle tick, Rhipicephalus (Boophilus) microplus infestations in cattle. In this study, we used the fact that these animals were naturally infected with Anaplasma marginale and Babesia bigemina to evaluate the effect of subolesin vaccination and gene knockdown on tick infection by these cattle tick-transmitted pathogens. Ticks fed on vaccinated cattle had lower subolesin mRNA levels when compared to controls, resembling RNAi results. A. marginale and B. bigemina infection was determined by PCR and decreased by 98% and 99%, respectively in ticks fed on vaccinated cattle and by 97% and 99%, respectively after subolesin knockdown. These results demonstrated that targeting subolesin expression by vaccination or RNAi results in lower subolesin mRNA and pathogen infection levels, probably due to the effect of subolesin downregulation on tick feeding, gene expression and gut and salivary glands tissue development and function. These results suggested that subolesin vaccines could be used for the dual control of tick infestations and pathogen infection, a result that could be relevant for other vectors and vector-borne pathogens.     

Vaccination with proteins involved in tick–pathogen interactions reduces vector infestations and pathogen infection

Tick-borne pathogens cause diseases that greatly impact animal health and production worldwide. The ultimate goal of tick vaccines is to protect against tick-borne diseases through the control of vector infestations and reducing pathogen infection and transmission. Tick genetic traits are involved in vector–pathogen interactions and some of these molecules such as Subolesin (SUB) have been shown to protect against vector infestations and pathogen infection. Based on these premises, herein we characterized the efficacy of cattle vaccination with tick proteins involved in vector–pathogen interactions, TROSPA, SILK, and Q38 for the control of cattle tick, Rhipicephalus (Boophilus) microplus infestations and infection with Anaplasma marginale and Babesia bigemina. SUB and adjuvant/saline placebo were used as positive and negative controls, respectively. The results showed that vaccination with Q38, SILK and SUB reduced tick infestations and oviposition with vaccine efficacies of 75% (Q38), 62% (SILK) and 60% (SUB) with respect to ticks fed on placebo control cattle. Vaccination with TROSPA did not have a significant effect on any of the tick parameters analyzed. The results also showed that vaccination with Q38, TROSPA and SUB reduced B. bigemina DNA levels in ticks while vaccination with SILK and SUB resulted in lower A. marginale DNA levels when compared to ticks fed on placebo control cattle. The positive correlation between antigen-specific antibody titers and reduction of tick infestations and pathogen infection strongly suggested that the effect of the vaccine was the result of the antibody response in vaccinated cattle. Vaccination and co-infection with A. marginale and B. bigemina also affected the expression of genes encoding for vaccine antigens in ticks fed on cattle. These results showed that vaccines using tick proteins involved in vector–pathogen interactions could be used for the dual control of tick infestations and pathogen infection.     

Mapping protective epitopes in the tick and mosquito subolesin ortholog proteins

The tick protective antigen, subolesin, is a structural and functional ortholog of insect akirins, an evolutionary conserved group of proteins that regulate gene expression thus affecting multiple cellular processes such as digestion, immune response, reproduction and development. In this study, we searched for common protective epitopes in tick and mosquito subolesin ortolog proteins. By combining the results of peptide and phage-display libraries scan analysis with sera from protected animals with computational modeling, three different epitope types (i) linear B-cell epitopes, (ii) conformational epitopes, and (iii) conformational discontinuous epitopes were identified. The determination of conserved protective epitopes in subolesin ortologs may lead to the development of a multi-target universal vaccine directed at the control of both arthropod infestations and reduction of vector capacity to transmit pathogens.     

Characterization of Aedes albopictus akirin for the control of mosquito and sand fly infestations

The control of arthropod vectors of pathogens that affect human and animal health is important for the eradication of vector-borne diseases. Recent evidences showed a reduction in the survival and/or fertility of mosquitoes, sand flies and poultry red mites fed in vitro with antibodies against the recombinant Aedes albopictus akirin. These experiments were the first step toward the development of a multi-target arthropod vaccine. In this study, we showed that the oviposition of A. albopictus and Phlebotomus perniciosus fed on mice vaccinated with recombinant A. albopictus akirin was reduced by 17% and 31%, respectively when compared to controls. However, Aedes aegypti mosquitoes were not affected after feeding on vaccinated mice. These results showed that recombinant A. albopictus akirin could be used to vaccinate hosts for the control of mosquito and sand fly infestations and suggested new experiments to develop improved vaccine formulations.     

Novel gene isolated from Caligus rogercresseyi: a promising target for vaccine development against sea lice

Sea lice (Copepoda, Caligidae) are the most widely distributed marine pathogens in the salmon industry in the last 30 years. Caligus rogercresseyi is the most important species affecting Chile's salmon industry. Vaccines against caligid copepods have the potential to be a cost-effective means of controlling the infestation and avoid many of the disadvantages of medicine treatments. However, research in the development of such vaccines has begun only recently and approaches used thus far have met with little or no success. In the present study, we characterized a novel gene (denoted as my32) from C. rogercresseyi which has the highest identity with the Lepeophtheirus salmonis gene akirin-2. To assess the function of the gene an RNA interference experiment was developed and a reduction in the number of ectoparasites on fish in the my32-dsRNA treated group was observed. The recombinant my32 protein was used in a vaccination-challenge trial to evaluate its ability to protect against sea lice infestations. A significant reduction in the number of parasites per fish was observed at 24 days post-challenge. These results, together with the delay observed in the development of parasites from the vaccinated group suggest that the major effect of immunization was on the second parasite generation. The results of these experiments suggest that the my32 protein may be a promising target for vaccine development to control sea lice infestations in fish.     

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.     

Control of Ixodes ricinus and Dermacentor reticulatus tick infestations in rabbits vaccinated with the Q38 Subolesin/Akirin chimera

Diseases transmitted by ticks greatly impact human and animal health and their control is important for the eradication of tick-borne diseases. Vaccination is an environmentally friendly alternative for tick control. Recent results have suggested that Subolesin/Akirin (SUB/AKR) are good candidate antigens for the control of arthropod vector infestations. Here, we describe the effect of vaccination with the Q38 chimera containing SUB/AKR conserved protective epitopes on Ixodes ricinus and Dermacentor reticulatus tick larval mortality, feeding and molting. We demonstrated that Q38 vaccination had an efficacy of 99.9% and 46.4% on the control of I. ricinus and D. reticulatus larvae by considering the cumulative effect on reducing tick survival and molting. The effect of the Q38 vaccine on larval feeding and molting is essential to reduce tick infestations and supports that Q38 might be a candidate universal antigen for the control of multiple tick species that can infest the same host.     

Control of tick infestations in wild roe deer (Capreolus capreolus) vaccinated with the Q38 Subolesin/Akirin chimera

Ticks (Acari: Ixodidae) are considered to be the most important vectors of disease-causing pathogens in domestic and wild animals, and emerging and re-emerging tick-borne diseases (TBD) exert an enormous impact on them. Wild ungulates are hosts for a wide variety of tick species and tick-borne pathogens that affect human and animal health. Consequently, the control of tick infestations and tick-borne pathogen prevalence is essential in some regions. Acaricides and animal management or culling have been used for the control of tick infestations and TBD, but tick vaccines constitute the best alternative to reduce the impact of acaricides on tick resistance and the environment. Previous results of controlled vaccination trials have shown that the Q38 Subolesin/Akirin chimera containing conserved protective epitopes could be a candidate universal antigen to control multiple tick species infestations. Thus, vaccination trials are necessary to validate these results under field conditions. In this study, we characterized the effect of Q38 vaccine on a wild population of European roe deer (Capreolus capreolus) in the Andalusian roe deer Reference Station (Junta de Andalucía, Cádiz, Spain). In this location, roe deer suffer especially severe parasitic conditions in some periods and commercial pesticides and ixodicides that are authorized to control ticks without specificity are frequently applied in the field, posing a threat to the environment. Animals vaccinated over a three-year period showed an antibody response to the vaccine antigen and a reduction in tick infestations by multiple species including Hyalomma marginatum, H. lusitanicum, Rhipicephalus bursa and Ixodes ricinus previously identified in roe deer, when compared to untreated controls. These results suggest the efficacy of Q38 for the control of tick infestations in wildlife.     

Vaccination with BM86, subolesin and akirin protective antigens for the control of tick infestations in white tailed deer and red deer

Red deer (Cervus elaphus) and white-tailed deer (Odocoileus virginianus) are hosts for different tick species and tick-borne pathogens and play a role in tick dispersal and maintenance in some regions. These factors stress the importance of controlling tick infestations in deer and several methods such as culling and acaricide treatment have been used. Tick vaccines are a cost-effective alternative for tick control that reduced cattle tick infestations and tick-borne pathogens prevalence while reducing the use of acaricides. Our hypothesis is that vaccination with vector protective antigens can be used for the control of tick infestations in deer. Herein, three experiments were conducted to characterize (1) the antibody response in red deer immunized with recombinant BM86, the antigen included in commercial tick vaccines, (2) the antibody response and control of cattle tick infestations in white-tailed deer immunized with recombinant BM86 or tick subolesin (SUB) and experimentally infested with Rhipicephalus (Boophilus) microplus, and (3) the antibody response and control of Hyalomma spp. and Rhipicephalus spp. field tick infestations in red deer immunized with mosquito akirin (AKR), the SUB ortholog and candidate protective antigen against different tick species and other ectoparasites. The results showed that deer produced an antibody response that correlated with the reduction in tick infestations and was similar to other hosts vaccinated previously with these antigens. The overall vaccine efficacy was similar between BM86 (E = 76%) and SUB (E = 83%) for the control of R. microplus infestations in white-tailed deer. The field trial in red deer showed a 25-33% (18-40% when only infested deer were considered) reduction in tick infestations, 14-20 weeks after the first immunization. These results demonstrated that vaccination with vector protective antigens could be used as an alternative method for the control of tick infestations in deer to reduce tick populations and dispersal in regions where deer are relevant hosts for these ectoparasites.