Protective efficacy of six recombinant proteins as vaccine candidates against Echinococcus granulosus in dogs

Background Cystic echinococcosis (CE) is caused by the infection of Echinococcus granulosus sensu lato (E. granulosus s.l.), one of the most harmful zoonotic helminths worldwide. Infected dogs are the major source of CE transmission. While praziquantel-based deworming is a main measure employed to control dog infections, its efficacy is at times compromised by the persistent high rate of dog re-infection and the copious discharge of E. granulosus eggs into the environment. Therefore, the dog vaccine is a welcome development, as it offers a substantial reduction in the biomass of E. granulosus. This study aimed to use previous insights into E. granulosus functional genes to further assess the protective efficacy of six recombinant proteins in dogs using a two-time injection vaccination strategy. Methods We expressed and combined recombinant E. granulosus triosephosphate isomerase (rEgTIM) with annexin B3 (rEgANXB3), adenylate kinase 1 (rEgADK1) with Echinococcus protoscolex calcium binding protein 1 (rEgEPC1), and fatty acid-binding protein (rEgFABP) with paramyosin (rEgA31). Beagle dogs received two subcutaneous vaccinations mixed with Quil-A adjuvant, and subsequently orally challenged with protoscoleces two weeks after booster vaccination. All dogs were sacrificed for counting and measuring E. granulosus tapeworms at 28 days post-infection, and the level of serum IgG was detected by ELISA. Results Dogs vaccinated with rEgTIM&rEgANXB3, rEgADK1&rEgEPC1, and rEgFABP-EgA31 protein groups exhibited significant protectiveness, with a worm reduction rate of 71%, 57%, and 67%, respectively, compared to the control group (P < 0.05). Additionally, the vaccinated groups exhibited an inhibition of worm growth, as evidenced by a reduction in body length and width (P < 0.05). Furthermore, the level of IgG in the vaccinated dogs was significantly higher than that of the control dogs (P < 0.05). Conclusion These verified candidates may be promising vaccines for the prevention of E. granulosus infection in dogs following two injections. The rEgTIM&rEgANXB3 co-administrated vaccine underscored the potential for the highest protective efficacy and superior protection stability for controlling E. granulosus infections in dogs.


Introduction
Cystic echinococcosis (CE) is a zoonotic parasitic disease caused by the ingestion of Echinococcus granulosus sensu lato (E.granulosus s.l.) eggs [1].CE poses a significant public health concern given its substantial morbidity in livestock and humans worldwide, most notably in remote areas that lack adequate hygiene and infrastructure [2,3].Recent estimates indicate that the worldwide prevalence of CE constitutes a considerable burden, with an annual new incidence of approximately 188,000 cases, thereby contributing to 1,097,000 disability-adjusted life years [4].In the life circle of E. granulosus, dogs play a crucial role as the major definitive hosts, serving as the main source of human CE infection [5].Thus, reducing E. granulosus infections in dogs holds great potential as a method for CE control.Currently, the implementation of praziquantel-based deworming has been a cornerstone measure to fight against E. granulosus infection in dogs [6,7].However, the deworming is often impractical because of the requirement frequency dosing (at least occur every 3 months) which is often not achieved in endemic areas [8,9].In Rio Negro and Morocco, prior studies have demonstrated that vaccinating the intermediate host with EG95 yielded an immune protection rate of 97%, whereas praziquantel treatment in owned dogs showed limited efficacy in reducing sheep incidence [10,11].Remarkably, analysis reveals that a dog deworming program lasting for many years in China, despite significant investment of human resources and financial means, has resulted in an escalation of overall infections of human CE [12].Thus, vaccination of dogs assumes a better substitution of deworming, as it can substantially reduce the parasite biomass within the dog intestine [13].Moreover, mathematical models indicates that a 75% reduction in worm burden through dog vaccination could potentially eliminate the transmission of E. granulosus to human and livestock [14].Additionally, considering that there is a much smaller population of dogs in comparison to intermediate hosts, dog vaccine would provide a more practical and feasible measure [6,15].
Initially, crude proteins were prepared from protoscoleces (PSCs) and cysts for the purpose of canine vaccination.However, the vaccination of dogs with such crude proteins has resulted in limited immune protection against E. granulosus infection [16].Recently, the development of E. granulosus vaccines has shifted towards utilizing recombinant subunit antigens expressed by bacterial systems which have now become the most used approaches [17].Among the vaccine candidates that have been thoroughly investigated, the recombinant proteins EgA31-EgTrp, EgM, and EgHCDH have exhibited promising potential in reducing worm burden and egg production [18][19][20].However, in the current phase of dog vaccine development, two obstacles impede the availability of licensed vaccines for field use.Firstly, the worm burdens in dogs of the vaccinated groups show massive variation.Secondly, in comparison to the twotime vaccine injection (including two doses for initial immunization and a booster per year) used for newborn lambs with EG95 (a licensed vaccine for sheep) [21], the current dog recombinant vaccines require three injections to achieve limited and unstable protection.This discrepancy in the number of required injections translates into a much higher financial cost.Given these challenges, it is crucial to enhance the stability of protection and optimize the immune protocol for dog vaccine against E. granulosus infection.
Our previous investigations revealed that the use of acquired recombinant proteins, including rEgTIM, rEgANXB3, rEgADK1, and rEgEPC1 [22][23][24][25], alongside the fused protein rEgFABP-EgA31, represent promising vaccine candidates.As previous studies, the use of two antigen components can enhance the protection stability of parasite vaccines [26].To further advance our research, we have introduced an innovative approach that involves the development of a two-protein combined vaccine (which applied two different systems, one with the coadministration of two proteins separately, and one with the administration of two fused proteins) administered via a two-time injection immunization protocol in dogs.In this study, we present our findings on the efficacy and immune response elicited by these vaccine candidates.

Ethics statement
The animal study was reviewed and approved by the Animal Care and Use Committee of Sichuan Agricultural University (SYXK2019-187).All animal procedures used in this study were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, Bethesda, MD, USA) and recommendations of the ARRIVE guidelines (https://www.nc3rs.org.uk/arrive-guidelines).All methods were carried out in accordance with relevant guidelines and regulations.

Animals and parasites
A total of 24 beagles, aged between four and five months, were obtained from the Beagles Breeding Center, Sichuan Institute of Musk Deer Breeding (Sichuan, China).The beagles consisted of an equal number of males and females, and all the dogs were dewormed with albendazole and praziquantel half month prior to the primary vaccination.Subsequently, the dogs were randomly assigned to their respective experimental groups.All the dogs were housed in Specific Pathogen Free conditions with a controlled quarantine facility and fed a standardized diet of commercial dog food with access to tap water.
PSCs were collected from the fresh livers with the hydatid cysts of sheep slaughtered in abattoir of Sichuan province, China.PSC viability and molecular genotyping was assessed using methods as described in our previous studies [27].PSCs with a viability higher than 95% and identified as E. granulosus sensu stricto (s.s.; G1-G3) (which were responsible for over 97% of CE infections in China [3]) were considered for the challenge process.
The PCR product was purified prior to digestion with restriction enzymes, ligated to the expression vector, pET-32a (Novagen, Madison, USA), and transformed into E. coli BL21 (DE3) (Cowin Biotech, Beijing, China).A positive E. coli clone with the correct DNA sequence was cultured in LB medium, and the expression of fused rEgFABP-EgA31 was induced using 1 mM IPTG at 16˚C for 10 h.The rEgFABP-EgA31 protein with a poly-histidine tag was affinity purified from bacterial lysates using the Ni-NTA His-tag resin (Qiagen, Hilden, Germany).The purified rEgFABP-EgA31 was analyzed by 12% SDS-PAGE, and the concentration of recombinant proteins was determined using a BCA protein assay kit (Beyotime, Jiangsu, China).

Vaccination and parasite challenge
The randomized allocation of the 24 Beagle dogs into four groups was conducted as described in Table 1 according to the requirements of World Association for the Advancement of Veterinary Parasitology [28].For each vaccination, 100 μg of each soluble recombinant protein and 1 mg Quil-A (Superfos Biosector, Denmark) in 1 mL PBS were mixed and left to stir overnight at 4˚C prior to administration.The dogs were administered a primary and booster vaccination via subcutaneous injection, with a 2-week interval between each vaccination.Two weeks after the booster vaccination, all dogs were orally challenged with 70,000 PSCs.Four weeks post- infection, all dogs were humanely euthanized and underwent necropsy (Fig 1).Tapeworms present in the small intestine of each dog were collected and recorded.The body length and width were also measured in 300 randomly selected tapeworms per group.The weekly sera extractions were performed for further IgG detection.

ELISA detection of serum IgG
ELISA conditions were optimized using a checkerboard titration method with the vaccine antigens and sera.The EgTIM, EgANXB3, EgADK1, EgEPC1, and EgFABP-EgA31 purified proteins were diluted in PBS to 5 μg/mL.The ELISA plates were coated with diluted antigen solutions and incubated overnight at 4˚C.The plates were subsequently washed three times with PBST and blocked with 5% skim milk for 2 h at 37˚C.After washing the wells three times, 100 μL of the serum samples (diluted 1:100 in PBST) were added and incubated at 37˚C for 1 h.The HRP-labeled rabbit anti-dog IgG (diluted 1:3000, Solarbio, Beijing, China) was added and incubated for 1.5 h at 37˚C.The wells were subsequently washed again and incubated with TMB (Tiangen, Beijing, China) substrate at 37˚C for 20 min.Finally, the reaction was stopped with 100 μL of 2 M H 2 SO 4 , and the optical density was measured at 450 nm.

Data collection and analysis
We employed a Mann-Whitney U test to statistically compare the worm burden in experimental groups with the control group.Data were analyzed with SPSS software v 22.0, and the average values were used to calculate the reduction in worm numbers.A P-value less than 0.05 was considered statistically significant.

Expression and purification of recombinant proteins
The recombinant protein preparations, including rEgANXB3, rEgTIM, rEgADK1, rEgEPC1, and fused rEgFABP-EgA31 were successfully expressed and purified.Our results demonstrated that the expressed proteins were of the expected molecular weights, with rEgANXB3 at approximately 53 kDa, rEgTIM at 46 kDa, rEgADK1 at 40 kDa, rEgEPC1 at 26 kDa, and the fused rEgFABP-EgA31 at 60 kDa, as confirmed by SDS-PAGE analysis.(Fig 2 ).

Vaccination of dogs with recombinant proteins
In comparison with the control group, the vaccination groups induced significant protection of the reduction in worm burdens and inhibition of worm growth, as detailed in Table 2.In terms of worm reduction, our results showed that the rEgTIM&rEgANXB3, rEgADK1&rE-gEPC1, and rEgFABP-rEgA31 vaccine groups exhibited superior protective efficacy, with a remarkable 71% (P = 0.004), 57% (P = 0.006), and 67% (P = 0.004) reduction in the worm burden after two subcutaneous injections, compared to the control group.As for the inhibition of worm growth, our results revealed that the worm growth of vaccine groups was significantly suppressed in 24% to 32% (P < 0.05) of the length and width of the worm bodies (Fig 3 and S1 Table ).

Dog serum IgG titers detection by ELISA
Our findings revealed that the experimental groups exhibited a remarkable increase in IgG titers compared to the control group (Fig 4).Notably, the highest levels of IgG were observed during the first week following the second booster vaccination, indicating a robust and prompt immune response.Furthermore, the IgG levels maintained a steady plateau within a certain range over time for weeks.However, the duration of the immune response had not yet been determined.

Discussion
In this study, we employed a two-protein combined formulation, which was administered to dogs using a two-time injection immunization protocol to evaluate the protectiveness of six vaccine candidates against E. granulosus s.s.infections.To the best of our knowledge, this is the first trial conducted in dogs that has evaluated this immunization strategy in the context of dog vaccine against E. granulosus s.s.tapeworms.Based on our previous studies and those of others, six proteins (rEgTIM, rEgANXB3, rEgADK1, rEgEPC1, rEgFABP and rEgA31) that have been demonstrated to play a crucial role in the development and survival of E. granulosus were selected as potential vaccine candidates [22][23][24][25].EgTIM is a pivotal enzyme in glycolysis, serving as the primary energy source for E. granulosus, and also known to be involved in several immune regulation functions [22,29].In addition, we found that annexins of E. granulosus s.s.might play an important role in parasitehost interactions and demonstrated that the level of EgANXB3 transcription was increased from PSCs to adults [23,30].This indicates that it has a potential role during the adult stages of infection in dogs.EgADK1 is a crucial enzyme involved in the regulation of adenosine triphosphate metabolism in E. granulosus.EgADK1 has been suggested as a potential vaccine candidate due to its role in cellular energy balance [24,31,32].Furthermore, EgEPC1 was selected as a pattern protein due to its robust immunogenicity and ability to interact with the host through secretion [33].E. granulosus EgA31 and EgFABP were previously identified as potential vaccine candidates [34,35].Specifically, recombinant EgA31 has been shown to enhance the cell-mediated immune response in dogs, indicating its potential as a promising vaccine antigen [34].Tapeworms are incapable of synthesizing fatty acids, and instead rely on acquiring fats from the host through the fatty acid binding protein [35].Based on these collective results, we hypothesized that the inclusion of rEgTIM&rEgANXB3, rEgADK1&rEgEPC1, and rEgFABP-EgA31, which had not been previously evaluated, might enhance the immune response and facilitate deworming efforts in the vaccinated group.Adjuvants play a critical role in vaccine development by enhancing antigen-specific immune responses [36].Out of several adjuvants (i.e., Quil-A, Freund's adjuvant, and ISCOMs) commonly employed in canine vaccines [17], Quil-A has demonstrated similar protective efficacy to that of antigens mixed with Freund's adjuvant or ISCOMs [37].By contrast, Quil-A is comparatively more cost-effective, stable, and safe compared to that of other adjuvants.Thus, in the present study, we opted to employ Quil-A emulsified with vaccine antigens to immunize dogs, and no adverse effects were observed among the subjects.Our findings suggested that the Quil-A adjuvant might represent a safe option for canine vaccines.
The serum IgG response was likely associated with the host protection against infection [37].Our data revealed that these recombinant proteins were successful in stimulating and sustaining the immune response in dogs over a period of weeks following primary vaccination.Meanwhile, our findings demonstrated that each of the three vaccine antigen groups significantly enhanced the protective immunity of dogs against E. granulosus infection, as evidenced by a reduction in worm burden and growth.
Compared to the current dog vaccine candidates, including rEgMs (with worm reduction rate 33%-99%), rEgHCDH (87%, not significant for P = 0.400) (administered via three subcutaneous injections) [19,37], and the Salmonella oral vaccine EgA31-EgTrp (70%-80%) [20], the vaccine candidates evaluated in our study demonstrated a favorable protective efficacy after two subcutaneous injections.Our findings revealed several advantages in terms of vaccine candidates: (a) the vaccination protocol with a reduction of one injection compared to existing   limitations were observed.The worm burdens in dogs of the vaccinated groups showed massive variation.However, compared to single antigen vaccines, such as rEgM and rEgHCDH, the data showed a smaller standard deviation, that suggested the two antigen vaccines might demonstrate better protection stability against parasite infection.Overall, our innovative strategy of combining two recombination proteins via two subcutaneous injections in dogs presented a highly practical and cost-effective solution for the development of canine vaccines against E. granulosus tapeworms.

Conclusions
Our findings suggested that this immunization protocol (two-protein combined vaccine administered via a two-time injection) could induce effective immune protection against E. granulosus infections in dogs.The rEgTIM&rEgANXB3 provided 71% protection against E. granulosus infection in dogs and exhibited superior protection stability, with a minimal standard deviation.Therefore, our research provided potential candidate vaccines and cost-effective immune protocol against E. granulosus infection.

Fig 3 .
Fig 3. E. granulosus worm burdens and sizes on day 28 post-infection in dogs.(A and B) The violin plot for the length and width of 300 worms in each group (***P < 0.001).(C) The size of each group of tapeworms under the microscope (×100).Representative images of the tapeworms in the control group, rEgTIM&rEgANXB3 group, rEgADK1&rEgEPC1 group, and rEgFABP-EgA31 group.https://doi.org/10.1371/journal.pntd.0011709.g003

Table 2 . E. granulosus worm burdens on day 28 post-infection in dogs vaccinated with vaccines mixed with Quil-A.
a Reduction (%) = (average worm number in control group-average worm number in the experiment group) / average worm number in control group × 100%.https://doi.org/10.1371/journal.pntd.0011709.t002

Table. The detailed data for length and width of E. granulosus s.s. worms at day 28 post- infection in dogs of vaccination and control groups
. Reduction (%) = (average worm size in control group-average worm size in the experiment group) / average worm size in control group × 100. (XLSX)