Codon optimization of voraxin α sequence enhances the immunogenicity of a recombinant vaccine against Hyalomma anatolicum infestation in rabbits

Codon optimization of voraxin α sequence enhances the immunogenicity of a recombinant vaccine against Hyalomma anatolicum infestation in rabbits | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Codon optimization of voraxin α sequence enhances the immunogenicity of a recombinant vaccine against Hyalomma anatolicum infestation in rabbits Mahmood Nazari, Zohre Monjezi,, Hedaiat allah Rooshanfekr, Fatemeh Salabi, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3865639/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Research has shown that voraxin α derived from male ticks stimulates blood feeding to engorge in female ticks. Whereas, the oviposition rate, egg weight, and body weight of female ticks were reduced in animals vaccinated with recombinant (r-) voraxin α. These data suggest a potential role of r-voraxin α as a functional anti-tick antigen in Rhipicephalus appendiculatus and Amblyomma hebraeum tick infestation. This study investigated the immunogenicity of r-voraxin α protein from Hyalomma anatolicum ( H. anatolicum ) tick as an anti-tick vaccine in rabbits. The H. anatolicum voraxin α sequence was optimized according to the codon usage in E. coli before being sub-cloned into pQE30. The gene sequence of the voraxin α was synthesized, verified by DNA sequencing, cloned in a pQE30 vector, and transformed into E. coli. Then, the expression of the r-voraxin α protein was confirmed by SDS-PAGE and Western blot analysis. Subsequently, three rabbits were immunized with the r-voraxin α as the vaccinated group, whereas three rabbits without injection were considered the control group. The result indicated the success of cloning of codon-optimized H. anatolicum voraxin α gene. Moreover, the expression of the r-voraxin α protein (approximately 18 kDa) in the bacterial expression system was confirmed by SDS-PAGE and Western blot analysis. The results of this study showed that the mortality rate in vaccine recipients increased compared to the control group ( P < 0.01 ). Also, the egg weight, oviposition rate, and engorgement weight of female ticks fed from vaccinated animals were significantly reduced compared to the control group ( P < 0.01 ). The results confirmed that the codon-optimized H. anatolicum voraxin α gene expressed in the bacterial expression system could be a suitable anti-tick vaccine against H. anatolicum tick infestation. Hyalomma anatolicum Ticks Voraxin α vaccine Codon optimization Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Ticks are responsible as vectors for transmitting pathogenic fungi, protozoa, viruses, and rickettsia in animals (Waladde et al., 1991). Ticks transmit pathogens to humans and play an important role in the process of disease transmission (Tick-borne diseases). Losses caused by tick infestation include weight loss, skin damage, and reduced milk production in the livestock industry (Sonenshine, 1991). Ticks are the main disease vectors for humans and domestic animals (Parola and Raoult, 2001; Peter et al., 2005). It is noteworthy that ticks and pathogens caused by them have a negative effect on cattle production (Graf et al. 2004; De la Fuente and Kocan 2003) .Ticks suck significant amounts of blood, which has deleterious effects on milk and meat production (Graf et al. 2004) as well as on livestock health (De la Fuente and Kocan 2003). It should be considered that annual economic losses worldwide are estimated at hundreds of millions of dollars due to the direct effects of tick infestation and diseases caused by tick pathogens (Peter et al., 2005). Hyalomma anatolicum ( H. anatolicum ) is one of the main infecting ticks in important livestock breeding areas that acts as a vector transmitting tick-borne diseases of both animals and humans (Biglari et al. 2018). The hard tick species such as H. anatolicum belong to the taxa Parasitiformes, Ixodoidea, and Ixodidae. The H. anatolicum causes serious damage to the public health and livestock industry. These ticks parasitize sheep, goats, cattle, camels, horses, and wild animals (Luo et al. 2021). Today, tick control is usually done using acaricides. Since using acaricides has many disadvantages, including chemical contamination of the environment and food (Miller et al. 2002), focusing on introducing vaccine candidates is a logical and central approach to controlling these ticks. The anti-tick vaccine is a generic approach for controlling ticks, which protects host animals from tick infestation. A large number of antigens including BM86 (Rand et al., 1989) BM95 (García-García et al., 2000) Serpins (Imamura et al., 2005) Subolesin and Ubiquitin (Almazan et al., 2010) etc. have been considered as vaccines against ticks infestation. Recently, the tick protective antigen of voraxin α has been suggested as a good candidate, which has promising results in controlling ixodid tick ( Rhipicephalus appendiculatus ) infestations (Yamada et al. 2009). Studies showed that voraxin α is an effective male pheromone in feeding and reproductive behavior (Kaufman, 2007). Injection of voraxin α into virgin females stimulates the blood-feeding to repletion in Amblyomma hebraeum , so that the mean weight of females fed on immunized rabbits showed a 72% reduction compared with the normal females fed on a control rabbit (Weiss and Kaufman, 2004). Moreover, the egg weight and engorgement weights of R. appendiculatus female ticks fed on immunized rabbits with voraxin α reduced by 50% and 40%, respectively (Yamada et al., 2009). Isolation, characterization, and molecular cloning of Voraxin α from Rhipicephalus appendiculatus (Yamada et al., 2009), Amblyomma hebraeum (Weiss and Kaufman, 2004), Rhipicephalus microplus (Kumar and Ghosh, 2016), and Hyalomma anatolicum (Nazari et al. 2022) were reported. In a previous study, we showed that voraxin α is expressed in the testis of H. anatolicum male ticks. Moreover, a voraxin α gene was sequenced from H. anatolicum ticks, and its characteristics were investigated in silico methods (Nazari et al. 2022). In this study, the voraxin α from H. anatolicum tick was cloned in a bacterial expression system, and then the immunogenicity of recombinant voraxin α protein as an anti-tick vaccine in rabbits was investigated. 2. Methods 2.1. Animals and ethical statement In this experimental study, six male adult New Zealand white rabbits weighing 2 ± 0.2 kg were housed in the Animal Housing Facility of Razi Vaccine and Serum Research Institute. All rabbits were acclimatized to new conditions for two weeks before the beginning of the experiment. Rabbits had free access to tap water and a commercial pellet diet. All animals received sufficient care according to “Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH publication No. 86-23, revised 1985). This investigation was approved by the local Ethics Committee in the Faculty of Veterinary Medicine (EE/1401.2.24.225571). 2.2. Codon Optimization and Synthetization The sequence of H. anatolicum voraxin α was previously presented by Nazari et al. (2022) (Accession Number GenBank: MZ356163) in The National Center for Biotechnology Information (NCBI) server. The 459-bp fragment of voraxin α ORF was optimized regarding E. coli as it is a popular host, synthesized chemically, and inserted into the expression vector pQE30 with Bam HI and Hind III restriction sites (ProteoGenix Co, France). 2.3. Cell transformation and Plasmid extraction The ordered pQE-30 plasmid, with an N-terminal hexa-histidine tag, transformed into competent E. coli BL21 (DE3) cells. The competent E. coli cells were prepared with Calcium Chloride according to previous protocols (Sambrook and Green 2012). The transformation was performed by the heat shock method. E. coli cells were cultivated on a TSB agar medium containing ampicillin. After selecting the recombinant clones, the plasmid DNA was extracted from the overnight cultured cells using the Miniprep plasmid isolation kit (SinaClon Company, Iran) according to the manufacturer’s instructions. Plasmid extraction was confirmed by agarose gel electrophoresis. The transformation accuracy was verified by a polymerase chain reaction and double digestion of the plasmid with BamHI and HindIII (Fermentas) enzymes, followed by DNA sequencing utilizing T5 primers. 2.4. Protein expression A colony of recombinant E.coli BL21 (DE3) cells, harboring the pQE30-voraxin α construct, was grown in TSB medium containing ampicillin (100 μg/ml) overnight at 37 °C with shaking at 120 rpm. Then, 4 ml of the overnight culture was inoculated to 100 ml fresh TSB, containing 100 μg/ml Ampicilin, and placed at 37 o C with shaking until the OD600 achieved to 0.6. Next, the voraxinα protein expression was induced by adding one mM isopropyl β-D-1- thiogalactopyranoside (IPTG, Sigma, USA), followed by incubation at 37 o C for four hours with shaking at 120 rpm. 2.5. Protein purification and Validation The r-voraxin α protein was purified from cellular extraction under denaturing conditions using the Ni-IDA column (Parstous, Iran). Briefly, the resin was washed with 25 ml of 0.15 M NaCl solution. Beads were equilibrated with 25 ml of the binding buffer (0.15 M NaCl, 10% glycerol, 0.5% Triton X-100, 50 Mm sodium phosphate buffer PH= 7.8). Once the resin has been washed and equilibrated, the protein sample is applied to the column. Then, the beads were washed again with 25 ml of the binding buffer. After that, the beads were washed with 25 ml of the wash buffer (Sodium phosphate buffer pH 7.8 (50 mM), 0.15 M NaCl with 3 mM Imidazole). This step will wash off any non-specific proteins bound to the beads and increase the purity of the final product. Finally, the protein was eluted (Recommended elution buffer consist of 0.1 M imidazole, 0.15 M NaCl with Sodium phosphate buffer pH= 7.8). The protein concentration was measured by Bradford assay (Bradford, M. M. 1976). The presence of r-voraxin α protein was evaluated by 12% SDS-PAGE. The purified protein (20 μg) was dialyzed through a dialysis membrane (10 kDa cutoff) to remove small peptides and change the buffer. To perform western blot analysis, after protein electrophoresis using the SDS-PAGE method, the gel was placed in transfer buffer (25 mMTris, 192 mM Glycine, and 20% methanol) for 10 to 15 minutes. A sandwich consisting of 5 layers including two layers of sponge, two layers of Whatman paper on both sides, one layer of nitrocellulose paper, and one layer of gel was prepared. All the following compounds were immersed in the transfection buffer for 15 min from preparation. After making sure that there were no bubbles in the gel, the sandwich with the cassette base was placed in the blotting tank that was already filled with the transfer buffer. The transmission was done with 60 volts and for 105 minutes. After transfer, the paper was washed three times with PBS for 5 minutes each time. Blocking was done by blocking buffer overnight in the refrigerator at 4 C. After blocking, the nitrocellulose paper was washed three times with PBS for 5 minutes each time. Subsequently, the nitrocellulose paper was incubated with primary antibodies (His-Tag (D3I1O) XP® Rabbit mAb Antibody; cell signaling technology) prepared at a dilution of 1.2000 to 1.5000 in PBS buffer for one hour at room temperature and on a shaker at 65 rpm. After incubation, the paper was washed three times with PBS for 5 minutes each time. Incubation was performed for secondary antibodies (mouse anti-rabbit IgG-HRP: sc-2357) prepared at 1/2000 dilution in PBS buffer for one hour. Finally, two solutions of ECL kit (Abcam, 133408, USA) were combined in a ratio of a 1:1 mixture in the amount of 250 microliters and poured on nitrocellulose paper with a sampler 1000 and soaked the paper for one minute. All processes are performed in a dark room under a red light. The papers were placed in the plastic protective cassette containing the sensitive film, and the bands were developed in the ChemiDoc Imaging System (Bio-Rad, USA). Photosensitive papers were scanned using an Image Lab Touch Software (Bio-Rad, USA), and the band density was calculated. 2.6. Immunogenicity analysis of r-voraxin protein in rabbits Six male New Zealand white rabbits (1.5 – 2.0 kg, 9 – 10 weeks of age, Research and Production Complex Pasteur Institute of Iran) were used for the immunization experiments. Rabbits were randomly divided into two groups, with three animals in each group. Three rabbits were immunized with the r-voraxin as the vaccinated group, whereas three rabbits were immunized with PBS as the control. Approximately 100 μg of the r-voraxin or PBS emulsified in a 1:1 mixture with complete Freund’s adjuvant system (complete adjuvant for the first challenge and incomplete for the subsequent challenge; Razi Vaccine and Serum Research Institute, Iran) was injected three times at two-week intervals (0, 2, and 4 weeks) for each animal at each immunization. The immunizations were performed as a subcutaneous injection. The serum was collected two-week intervals (0, 2, and 4 weeks) before each injection to estimate the serum antibody titers. The serum antibody titers in vaccinated rabbits were analyzed by ELISA. 2.7. Serum titer estimation Serum antibody titers of vaccinated rabbits were performed by ELISA according to the method of Yamada et al. (2009). Polystyrene microtiter plates were coated with r-voraxin α (1 μg) solubilized in carbonate buffer and then washed three times with ELISA wash buffer (PBS containing 0.05% Tween20). The coated plates were incubated with immunized rabbit sera (diluted 1:100 in PBS). Plates were incubated at 37 ºC for 1 hour, and washed with ELISA wash buffer. A specific antibody was detected by horseradish peroxidase (HRP) conjugated protein A/G (Rahazist padtan, Iran) diluted 1:500 in PBST at 37 º C for one hour. After washing three times with PBST, TMB (3,3′,5,5′-Tetramethylbenzidine) was added and incubated for 10 minutes and stopped after fifteen minutes with 3 M sulfuric acid. Finally, the absorbance of each well was measured at 450nm. 2.8. Tick-challenge experiment Tick-challenge infestation was performed as previously described (Yamada et al. 2009). Three rabbits were immunized with the r-voraxin α as the vaccinated group, whereas three rabbits without injection were considered the control group. All rabbits were infested with six pairs of adult ticks trapped in ear bags two weeks after the last immunization. To analyze the immunogenic effect of the r-voraxin α on tick infestation, a visual examination was performed daily for up to 14 days. The measured parameters included attachment rates, mortality rates, feeding duration, engorgement weight, oviposition rate, and egg weight. 2.9. Statistics All data were expressed as mean ± SEM. Statistical significance was determined by Student’s t-test using SAS software (SAS Institute, 2013). 3. Results 3.1. Codon optimization and cloning According to ProteoGenix results (Additional File 1), CAI (Codon Adaptation Index) was estimated to be 0.68. Replacement of rare and less preferential codons with more favorable codon usage improved the expression level in E. coli. CAI was measured again to be 0.98 after codon optimization, which was within the optimum range. The comparison of GC content between the original sequence and the optimized sequence of the voraxin α is presented in Additional File 1. The Average GC content had a suitable value after (0.49) and before (0.58) optimizing. The codon-optimized of the voraxin α sequence was synthesized and sub-cloned into pQE-30 by ProteoGenix Company. In recognition of sub-cloning, recombinant pQE30 was digested by Bam HI and Hind III. Accuracy of sub-cloning was confirmed by sequencing (Additional File 2). The ordered pQE-30 plasmid was transformed into competent E. coli cells and sequenced for final confirmation after plasmid extraction (Additional File 3). 3.2. In vitro protein expression and western blot analysis As shown in Fig. 1, the r-voraxin protein made in the pQE30 vector (considering the presence of 6x His tag on the N-terminus) was expressed as an 18 kDa protein on a 12% polyacrylamide gel, consistent with the predicted molecular mass. Ni-IDA Purification System was used to purify the recombinant protein carrying a 6x His tag. After the purification, the r-voraxin α protein was solubilized in PBS, and used as antigen for western blot analysis. Western blot analysis was performed on protein extracts to detect the r-voraxin α protein weight and their properties. The r-voraxin α protein appeared as a single band with a molecular weight of 18 kDa (Fig. 2). These results indicated that the r-voraxin α protein was synthetized in E.coli BL21 (DE3) cells in vitro. The concentration of proteins was estimated to be about 3.1 mg.mL-1. 3.3. Serum antibody titers The humoral response, three times at two-week intervals (0, 2, and 4 weeks), was detected by ELISA on serum from immunized and control rabbits before the tick challenge. As shown in Fig. 3, the IgG titer on the serum of the immunized rabbits significantly increased compared to the control, reaching 0.284 and 0.465 (mean value of three rabbits) in the second and fourth weeks, respectively. 3.4. Vaccination trials Seven, fourteen, and twenty-one days after the final immunization, sera obtained from immunized rabbits reacted with the r-voraxin α antigen by Western blot analysis during the tick challenge (Fig 4). Western blot results confirmed the presence of an 18 kDa band for the r-voraxin α antigen. As shown in Fig. 4, the r-voraxin α antigen decreased over a 21-day time course. 3.5. Effects of the r-voraxin α vaccine on tick feeding The biological parameters included attachment rates, mortality rates, feeding duration, engorgement weight, oviposition rate, and egg weight measured to assess the immunogenic effect of the r-voraxin α on female ticks during 14 days (Table 1). There was no observed difference between the vaccinated groups (84.0%) in comparison with the control group (85.0%) (Measured on day 5). Also, there was no apparent difference in the feeding duration between vaccinated and control rabbits. The mortality rate in the vaccinated group increased compared to the control group (P < 0.01). In brief, 18/30 female ticks (60% of ticks fed on vaccinated rabbits) and 5/30 female ticks (16.6% of ticks fed on control rabbits) died during the vaccination trial. According to the data presented in Table 1, the weight of eggs and the body weight of engorged female ticks fed on vaccinated animals was significantly reduced compared to the control group ( P < 0.01). Also, the oviposition rate parameter was significantly decreased by 41.6% (5/12 ticks) in female ticks fed on vaccinated rabbits. While almost all live ticks (24/25 ticks) isolated from the control rabbits group laid live eggs. Table 1. Effects of the r-voraxin vaccine α on H. anatolicum Tick feeding Immunized group a Control Vaccinated (r-voraxin) 85.0 84.0 Attachment rates (%) 8.5 ± 0.75 (25) 7.5 ±0.40 (12) Feeding duration (days) of female tick (n) 613.0 ± 23.1 (25) 159.0 ± 25.7 ** (12) Fed weight (mg) of female tick (n) 295.4 ± 9.5 (25) 54.4 ± 5.6 ** (12) Weight (mg) of egg mass (n) 16.6 60 Mortality rate b (%) 96 c 58.4 Oviposition rate (%) a) Each group contains three rabbits. Six pairs of adult ticks were introduced into each ear bag (right and left). Results are expressed as the mean ± SEM for each ear bag. b) Mortality rate was obtained from the ticks that could not survive during feeding. c) P < 0.01 compared to the control (two-tailed Student’s t-test) 4. Discussion Although H. anatolicum is recognized as one of the main infesting ticks in important livestock breeding areas, more effective anti-tick vaccines for H. anatolicum are not yet available. Therefore, more studies are needed to identify candidate antigens for developing anti-tick vaccines against H. anatolicum . Previous studies have shown that male tick-derived voraxinα from Amblyomma hebraeum tick is essential for female engorgement and oviposition (Weiss and Kaufman, 2004). The biological parameters included the oviposition rate, egg weight, and body weight of female ticks decreased in animals vaccinated with r-voraxin α of Rhipicephalus appendiculatus (Yamada et al. 2009). These results have shown the possibility of r-voraxin α as a functional anti-tick antigen in Rhipicephalus appendiculatus . Recently, molecular cloning, characterization, and homologs of voraxin α were reported in H. anatolicum (Nazari et al. 2022). In this report, the expression of the voraxin α protein was evaluated in the bacterial expression system as well as the immunogenicity effect of the r-voraxin α antigen in H. anatolicum infestation as an anti-tick vaccine. Since rare codons affect the expression level of recombinant proteins in the bacterial expression system, therefore codon optimization with preferred codons can increase the expression level in the host (Aghaei et al. 2021) and facilitate protein folding (Dali et al. 2011). For this purpose, the bacterial system was used for protein expression, and the codons of voraxin α were optimized. According to the results, the CAI showed a suitable and significant range for high expression after optimizing. CAI was measured to be 0.98 after codon optimization, which was within the optimum range. A CAI of 1.0 is considered perfect, while a CAI greater than 0.8 is rated suitable for expression in the desired expression organism. Therefore, there is a greater chance of protein expression at a high level (Carbone 2003). Therefore, the efficiency of voraxin α production increased by using an optimized codon. The Average GC content had a suitable value after optimization. The ideal percentage range of GC content is between 30 and 70%. The values outside of this range will affect the efficiency of transcription (Puigbo 2007). The recombinant protein was purified by Ni2 + affinity chromatography and then confirmed by SDS-PAGE and western blot analysis. Recently, reports indicated that these tags do not affect the efficiency of r-voraxin α. In addition, similar results were observed regarding the molecular weight (18 kDa) of r-voraxinα expressed in E. coli Yamada et al. 2009; Weiss and Kaufman, 2004). In the vaccination trial, r-voraxin α induced humoral immune response in immunized rabbits, as determined by ELISA, suggesting that rabbit antibodies elicited upon vaccination could react with the r-voraxin α protein. Consistent with this research, previous studies indicated that the immunization trial increased the mortality rate and reduced the egg weight, oviposition rate, and engorgement weight of female ticks upon challenge infestation of H. anatolicum in rabbits (Yamada et al. 2009; Weiss and Kaufman, 2004). The mortality rate obtained in this research (60%) was higher than the mortality rate reported by Yamada (26.7%) (Yamada et al. 2009). Reduction in the egg weight and engorgement weight of female ticks showed in this study were approximately 75% and 81%, whereas the egg weight and engorgement weight of female ticks reported by Yamada et al. (2009) were 50% and 40%, respectively. Weiss and Kaufman (2004) reported that the mean weight of females fed on immunized rabbits showed a 72% reduction compared with the normal females fed on control rabbits infected with Amblyomma hebraeum tick. In all previous reports, the voraxin α was expressed without codon optimization. The Amblyomma hebraeum voraxin α was expressed in the Sf21 insect cell line (Weiss and Kaufman 2004). Also, the R. appendiculatus voraxin α expression was performed in E. coli strain Rosetta-gami DES LysS and Drosophila Schneider 2 (S2) insect cells without codon optimization. Although the expression occurred in both bacterial and insect cells, the results showed that the bacterial cell is more suitable (Yamada et al. 2009). The reason for the greater effectiveness of voraxin α may be related to the optimization of the voraxin α codon in this study. Codon optimization enhances protein expression and folding in the host. Even though there were no differences in the attachment rates between vaccinated and control groups, an apparent increase in the mortality rate indicated that vaccination of rabbits with the r-voraxin α had a protective effect against H. anatolicum ticks. 5. Conclusions Codons of H. anatolicum voraxin α sequence were optimized according to the codon usage in E. coli. A codon-optimized voraxin α was synthesized into a pQE30 expression vector. The r-voraxin α expression were successfully performed in E. coli. The r-voraxin α protein was purified from cellular extraction and appeared as a single band with a molecular weight of 18 kDa. The results of this research suggested that the codon-optimized H. anatolicum voraxin α sequence expressed in the bacterial expression system could be considered a vaccine with significant protective potential for immunized hosts against H. anatolicum tick infestation. Declarations Declaration of competing interest The authors confirm that there are no known conflicts of interest associated with this publication. Data availability The authors confirm that the data supporting the findings of this study are available within the article (and/or) its supplementary materials. Ethics We hereby declare, all ethical standards have been respected in preparation of the submitted article. 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Cloning and characterization of Rhipicephalus appendiculatus voraxin and its effect as anti-tick vaccine. Vaccine 2009; 27(43): 5989–5997. https://doi: 10.1016/j.vaccine.2009.07.072. Additional Declarations No competing interests reported. Supplementary Files AdditionalFile1.pdf AdditionalFile2.pdf AdditionalFile3.ab1 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3865639","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":267561446,"identity":"5133e2eb-ed15-44e7-9eb3-18a57ae4d55e","order_by":0,"name":"Mahmood Nazari","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIiWNgGAWjYBADHn4InZAAoYjRItlAqhYGgwPIWvAB3Qbegx9/1NyTMT5+/Nnjyra0PAb2ww8YHu7BrcXsAF+yhMSxYh6zMznmhmfbcooZeNIMGBKe4dPCYyBhwJbAY3Ygh02ysa0isYEhB+i+A3i1GP9I+JfAY9z//BlEC/8bglrMJA62JQDtSjADaslJbJAgZMthHjPLxr4EHokbb8wkG86lJbZJPDM4gFfL8R7jmz++Jdjz96c/k2woS07s509++PAHHi0MzOgCbECMT8MoGAWjYBSMAiIAADdjTkcaGqvcAAAAAElFTkSuQmCC","orcid":"","institution":"Agricultural Sciences and Natural Resources University of Khuzestan","correspondingAuthor":true,"prefix":"","firstName":"Mahmood","middleName":"","lastName":"Nazari","suffix":""},{"id":267561447,"identity":"8a8dc69a-3936-4105-9f3a-e1e38fababda","order_by":1,"name":"Zohre Monjezi,","email":"","orcid":"","institution":"Agricultural Sciences and Natural Resources University of Khuzestan","correspondingAuthor":false,"prefix":"","firstName":"","middleName":"Zohre","lastName":"Monjezi","suffix":""},{"id":267561448,"identity":"4fa7cfdb-e09d-4282-9d2a-1a7e22c5dc12","order_by":2,"name":"Hedaiat allah Rooshanfekr","email":"","orcid":"","institution":"Agricultural Sciences and Natural Resources University of Khuzestan","correspondingAuthor":false,"prefix":"","firstName":"Hedaiat","middleName":"allah","lastName":"Rooshanfekr","suffix":""},{"id":267561449,"identity":"2e0fe3a3-1395-477e-a2f4-b60d9ece06ae","order_by":3,"name":"Fatemeh Salabi","email":"","orcid":"","institution":"Razi Vaccine and Serum Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Fatemeh","middleName":"","lastName":"Salabi","suffix":""},{"id":267561450,"identity":"1f975e60-43e1-44ba-86c7-88458e50a892","order_by":4,"name":"Mohammad Reza Tabandeh","email":"","orcid":"","institution":"Shahid Chamran University of Ahvaz","correspondingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"Reza","lastName":"Tabandeh","suffix":""}],"badges":[],"createdAt":"2024-01-15 06:59:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3865639/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3865639/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49802853,"identity":"958d5385-02a9-4cc8-8f2a-501e4a61e15c","added_by":"auto","created_at":"2024-01-18 09:16:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":217167,"visible":true,"origin":"","legend":"\u003cp\u003eSDS-PAGE analysis of the r-voraxin α expressed in E.coli BL21 (DE3) cells. A: Negative control (Uninduced E. coli lysates) B: Purified induced E. coli lysates C: Unpurified induced E. coli lysates D: The 18 kDa r-voraxinα protein purified from Ni-IDA Resin column were electrophoresed on 12% polyacrylamide gel and stained with Coomassie Brilliant blue.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3865639/v1/43a594d772661da4f0cd1213.png"},{"id":49802856,"identity":"0dccbaac-a29f-4304-93f7-834cdf5d4650","added_by":"auto","created_at":"2024-01-18 09:16:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":35878,"visible":true,"origin":"","legend":"\u003cp\u003eWestern blot of the r-voraxin α protein (A: Western blot of the recombinant protein before purification; number B: Western blot after purification of the recombinant protein of voraxin α)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3865639/v1/e973a029d5af6ec9a223aa8e.png"},{"id":49802854,"identity":"d09c634a-e854-418d-aad3-732efdd311f0","added_by":"auto","created_at":"2024-01-18 09:16:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":21897,"visible":true,"origin":"","legend":"\u003cp\u003eAntibody response in immunized and control rabbits during immunization schedule by ELISA test\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3865639/v1/e449eb1498e2d3baba31f42a.png"},{"id":49802855,"identity":"6ad8b0d9-f78d-462f-ac25-8e4e9806e9bd","added_by":"auto","created_at":"2024-01-18 09:16:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":65669,"visible":true,"origin":"","legend":"\u003cp\u003eWestern blot results of sera obtained from rabbits immunized with the r-voraxin α over a 21-day course.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3865639/v1/727fa05c672071be13855b55.png"},{"id":56085293,"identity":"bba8c319-23ba-43ad-adc3-59ced8e96de9","added_by":"auto","created_at":"2024-05-08 10:51:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":819488,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3865639/v1/476d6075-a392-49e5-8e51-d74322aee1f0.pdf"},{"id":49802859,"identity":"3f75a73b-2129-4ab6-b453-643d557a2db7","added_by":"auto","created_at":"2024-01-18 09:16:35","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":372281,"visible":true,"origin":"","legend":"","description":"","filename":"AdditionalFile1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3865639/v1/845cf8fd2b92935c144307aa.pdf"},{"id":49802858,"identity":"a9842545-21a9-4e76-ad83-db4111f8bc24","added_by":"auto","created_at":"2024-01-18 09:16:35","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":156920,"visible":true,"origin":"","legend":"","description":"","filename":"AdditionalFile2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3865639/v1/9aa5f4964ee35147c84d781f.pdf"},{"id":49802860,"identity":"ad2d2d77-731d-4ff6-9618-63907f986202","added_by":"auto","created_at":"2024-01-18 09:16:35","extension":"ab1","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":269923,"visible":true,"origin":"","legend":"","description":"","filename":"AdditionalFile3.ab1","url":"https://assets-eu.researchsquare.com/files/rs-3865639/v1/619deb29559119834dbd31c3.ab1"}],"financialInterests":"No competing interests reported.","formattedTitle":"Codon optimization of voraxin α sequence enhances the immunogenicity of a recombinant vaccine against Hyalomma anatolicum infestation in rabbits","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eTicks are responsible as vectors for transmitting pathogenic fungi, protozoa, viruses, and rickettsia in animals (Waladde et al., 1991). Ticks transmit pathogens to humans and play an important role in the process of disease transmission (Tick-borne diseases). Losses caused by tick infestation include weight loss, skin damage, and reduced milk production in the livestock industry (Sonenshine, 1991). Ticks are the main disease vectors for humans and domestic animals (Parola and Raoult, 2001; Peter et al., 2005). It is noteworthy that ticks and pathogens caused by them have a negative effect on cattle production (Graf et al. 2004; De la Fuente and Kocan 2003) .Ticks suck significant amounts of blood, which has deleterious effects on milk and meat production (Graf et al. 2004) as well as on livestock health (De la Fuente and Kocan 2003). It should be considered that annual economic losses worldwide are estimated at hundreds of millions of dollars due to the direct effects of tick infestation and diseases caused by tick pathogens (Peter et al., 2005).\u003c/p\u003e \u003cp\u003e \u003cem\u003eHyalomma anatolicum\u003c/em\u003e (\u003cem\u003eH. anatolicum\u003c/em\u003e) is one of the main infecting ticks in important livestock breeding areas that acts as a vector transmitting tick-borne diseases of both animals and humans (Biglari et al. 2018). The hard tick species such as \u003cem\u003eH. anatolicum\u003c/em\u003e belong to the taxa Parasitiformes, Ixodoidea, and Ixodidae. The \u003cem\u003eH. anatolicum\u003c/em\u003e causes serious damage to the public health and livestock industry. These ticks parasitize sheep, goats, cattle, camels, horses, and wild animals (Luo et al. 2021).\u003c/p\u003e \u003cp\u003eToday, tick control is usually done using acaricides. Since using acaricides has many disadvantages, including chemical contamination of the environment and food (Miller et al. 2002), focusing on introducing vaccine candidates is a logical and central approach to controlling these ticks. The anti-tick vaccine is a generic approach for controlling ticks, which protects host animals from tick infestation.\u003c/p\u003e \u003cp\u003eA large number of antigens including BM86 (Rand et al., 1989) BM95 (Garc\u0026iacute;a-Garc\u0026iacute;a et al., 2000) Serpins (Imamura et al., 2005) Subolesin and Ubiquitin (Almazan et al., 2010) etc. have been considered as vaccines against ticks infestation. Recently, the tick protective antigen of voraxin α has been suggested as a good candidate, which has promising results in controlling \u003cem\u003eixodid\u003c/em\u003e tick (\u003cem\u003eRhipicephalus appendiculatus\u003c/em\u003e) infestations (Yamada et al. 2009). Studies showed that voraxin α is an effective male pheromone in feeding and reproductive behavior (Kaufman, 2007). Injection of voraxin α into virgin females stimulates the blood-feeding to repletion in \u003cem\u003eAmblyomma hebraeum\u003c/em\u003e, so that the mean weight of females fed on immunized rabbits showed a 72% reduction compared with the normal females fed on a control rabbit (Weiss and Kaufman, 2004). Moreover, the egg weight and engorgement weights of \u003cem\u003eR. appendiculatus\u003c/em\u003e female ticks fed on immunized rabbits with voraxin α reduced by 50% and 40%, respectively (Yamada et al., 2009). Isolation, characterization, and molecular cloning of Voraxin α from \u003cem\u003eRhipicephalus appendiculatus\u003c/em\u003e (Yamada et al., 2009), \u003cem\u003eAmblyomma hebraeum\u003c/em\u003e (Weiss and Kaufman, 2004), \u003cem\u003eRhipicephalus microplus\u003c/em\u003e (Kumar and Ghosh, 2016), and \u003cem\u003eHyalomma anatolicum\u003c/em\u003e (Nazari et al. 2022) were reported. In a previous study, we showed that voraxin α is expressed in the testis of \u003cem\u003eH. anatolicum\u003c/em\u003e male ticks. Moreover, a voraxin α gene was sequenced from \u003cem\u003eH. anatolicum\u003c/em\u003e ticks, and its characteristics were investigated \u003cem\u003ein silico\u003c/em\u003e methods (Nazari et al. 2022). In this study, the voraxin α from \u003cem\u003eH. anatolicum\u003c/em\u003e tick was cloned in a bacterial expression system, and then the immunogenicity of recombinant voraxin α protein as an anti-tick vaccine in rabbits was investigated.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cp\u003e2.1. \u003cstrong\u003eAnimals and ethical statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this experimental study, six male adult New Zealand white rabbits weighing 2 ± 0.2 kg were housed in the Animal Housing Facility of Razi Vaccine and Serum Research Institute. All rabbits were acclimatized to new conditions for two weeks before the beginning of the experiment. Rabbits had free access to tap water and a commercial pellet diet. All animals received sufficient care according to “Guide for the Care and Use of Laboratory Animals\" published by the National Institutes of Health (NIH publication No. 86-23, revised 1985). This investigation was approved by the local Ethics Committee in the Faculty of Veterinary Medicine (EE/1401.2.24.225571).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2. Codon Optimization and Synthetization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sequence of \u003cem\u003eH. anatolicum\u003c/em\u003e voraxin\u0026nbsp;α\u0026nbsp;was previously presented by Nazari et al. (2022) (Accession Number GenBank: MZ356163) in The National Center for Biotechnology Information (NCBI) server. The 459-bp fragment of voraxin\u0026nbsp;α\u0026nbsp;ORF was optimized regarding E. coli as it is a popular host, synthesized chemically, and inserted into the expression vector pQE30 with Bam HI and Hind III restriction sites (ProteoGenix Co, France).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3. Cell transformation and Plasmid extraction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ordered pQE-30 plasmid, with an N-terminal hexa-histidine tag, transformed into competent E. coli BL21 (DE3) cells. The competent E. coli cells were prepared with Calcium Chloride according to previous protocols (Sambrook\u0026nbsp;and Green 2012). The transformation was performed by the heat shock method. E. coli cells were cultivated on a TSB agar medium containing ampicillin. After selecting the recombinant clones, the plasmid DNA was extracted from the overnight cultured cells using the Miniprep plasmid isolation kit (SinaClon Company, Iran) according to the manufacturer’s instructions. Plasmid extraction was confirmed by agarose gel electrophoresis. The transformation accuracy was verified by a polymerase chain reaction and double digestion of the plasmid with BamHI and HindIII (Fermentas) enzymes,\u0026nbsp;followed by DNA sequencing utilizing T5 primers.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4. Protein expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA colony of recombinant E.coli BL21 (DE3) cells, harboring the pQE30-voraxin α construct, was grown in TSB medium containing ampicillin (100 μg/ml) overnight at 37 °C with shaking at 120 rpm. Then, 4 ml of the overnight culture was inoculated to 100 ml fresh TSB, containing 100 μg/ml Ampicilin, and placed at 37\u003csup\u003eo\u003c/sup\u003e C with shaking until the OD600 achieved to 0.6. Next, the voraxinα protein expression was induced by adding one mM isopropyl β-D-1- thiogalactopyranoside (IPTG, Sigma, USA), followed by incubation at 37\u003csup\u003eo\u003c/sup\u003eC for four hours with shaking at 120 rpm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5. Protein purification and Validation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe r-voraxin α protein was purified from cellular extraction under denaturing conditions using the Ni-IDA column (Parstous, Iran). Briefly,\u0026nbsp;the resin was washed with 25 ml of 0.15 M NaCl solution. Beads were equilibrated with 25 ml of the binding buffer (0.15 M NaCl, 10% glycerol, 0.5% Triton X-100, 50 Mm sodium phosphate buffer PH= 7.8). Once the resin has been washed and equilibrated, the protein sample is applied to the column. Then, the beads were washed again with 25 ml of the binding buffer. After that, the beads were washed with 25 ml of the wash buffer (Sodium phosphate buffer pH 7.8 (50 mM), 0.15 M NaCl with 3 mM Imidazole). This step will wash off any non-specific proteins bound to the beads and increase the purity of the final product. Finally, the protein was eluted (Recommended elution buffer consist of 0.1 M imidazole, 0.15 M NaCl with Sodium phosphate buffer pH= 7.8). The protein concentration was measured by Bradford assay\u0026nbsp;(Bradford, M. M. 1976). The presence of r-voraxin α protein was evaluated by 12% SDS-PAGE. The purified protein (20 μg) was dialyzed through a dialysis membrane (10 kDa cutoff) to remove small peptides and change the buffer.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo perform western blot analysis,\u0026nbsp;after protein electrophoresis using the SDS-PAGE method,\u0026nbsp;the gel was placed in transfer buffer (25 mMTris, 192 mM Glycine, and 20% methanol) for 10 to 15 minutes. A sandwich consisting of 5 layers including two layers of sponge, two layers of Whatman paper on both sides, one layer of nitrocellulose paper, and one layer of gel was prepared. All the following compounds were immersed in the transfection buffer for 15 min from preparation. After making sure that there were no bubbles in the gel, the sandwich with the cassette base was placed in the blotting tank that was already filled with the transfer buffer.\u0026nbsp;The transmission was done with 60 volts and for 105 minutes. After transfer, the paper was washed three times with PBS for 5 minutes each time. Blocking was done by blocking buffer overnight in the refrigerator at 4 C.\u0026nbsp;After blocking, the nitrocellulose paper was washed three times with PBS for 5 minutes each time. Subsequently, the nitrocellulose paper was incubated with primary antibodies (His-Tag (D3I1O) XP® Rabbit mAb Antibody; cell signaling technology) prepared at a dilution of 1.2000 to 1.5000 in PBS buffer for one hour at room temperature and on a shaker at 65 rpm. After incubation, the paper was washed three times with PBS for 5 minutes each time. Incubation was performed for secondary antibodies (mouse anti-rabbit IgG-HRP: sc-2357) prepared at 1/2000 dilution in PBS buffer for one hour. Finally, two solutions of ECL kit (Abcam, 133408, USA) were combined in a ratio of a 1:1 mixture in the amount of 250 microliters and poured on nitrocellulose paper with a sampler 1000 and soaked the paper for one minute. All processes are performed in a dark room under a red light. The papers were placed in the plastic protective cassette containing the sensitive film, and the bands were developed in\u0026nbsp;the ChemiDoc Imaging System\u0026nbsp;(Bio-Rad, USA). Photosensitive papers were scanned using an\u0026nbsp;Image Lab Touch Software\u0026nbsp;(Bio-Rad, USA), and the band density was calculated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6. Immunogenicity analysis of r-voraxin protein in rabbits\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSix male New Zealand white rabbits (1.5 – 2.0 kg, 9 – 10 weeks of age, Research and Production Complex Pasteur Institute of Iran) were used for the immunization experiments. Rabbits were randomly divided into two groups, with three animals in each group. Three rabbits were immunized with the r-voraxin as the vaccinated group, whereas three rabbits were immunized with PBS as the control. Approximately 100 μg of the r-voraxin or PBS emulsified in a 1:1 mixture with complete Freund’s adjuvant system (complete adjuvant for the first challenge and incomplete for the subsequent challenge; Razi Vaccine and Serum Research Institute, Iran) was injected three times at two-week intervals (0, 2, and 4 weeks) for each animal at each immunization. The immunizations were performed as a subcutaneous injection. The serum was collected two-week intervals (0, 2, and 4 weeks) before each injection to estimate the serum antibody titers. The serum antibody titers in vaccinated rabbits were analyzed by ELISA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7. Serum titer estimation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSerum antibody titers\u0026nbsp;of vaccinated rabbits were performed by ELISA\u0026nbsp;according to the method of\u0026nbsp;Yamada et al. (2009). Polystyrene\u0026nbsp;microtiter plates were coated with\u0026nbsp;r-voraxin α (1 μg)\u0026nbsp;solubilized in carbonate buffer and then washed three times with ELISA wash buffer (PBS containing 0.05% Tween20).\u0026nbsp;The coated plates were incubated with immunized rabbit sera (diluted 1:100 in PBS). Plates were incubated at 37 ºC for 1 hour, and washed with ELISA\u0026nbsp;wash buffer.\u0026nbsp;A specific antibody was detected by\u0026nbsp;horseradish peroxidase (HRP)\u0026nbsp;conjugated\u0026nbsp;protein A/G\u0026nbsp;(Rahazist padtan, Iran)\u0026nbsp;diluted 1:500 in PBST at\u0026nbsp;37 \u003csup\u003eº\u003c/sup\u003eC\u0026nbsp;for one hour. After washing three times with PBST, TMB (3,3′,5,5′-Tetramethylbenzidine) was added and incubated for 10 minutes and stopped after fifteen minutes with 3 M sulfuric acid. Finally, the absorbance of each well was measured at 450nm.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.8. Tick-challenge experiment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTick-challenge infestation was performed as previously described (Yamada et al. 2009). Three rabbits were immunized with the r-voraxin\u0026nbsp;α\u0026nbsp;as the vaccinated group, whereas three rabbits without injection were considered the control group. All rabbits were infested with six pairs of adult ticks trapped in ear bags two weeks after the last immunization. To analyze the immunogenic effect of the r-voraxin\u0026nbsp;α\u0026nbsp;on tick infestation, a visual examination was performed daily for up to 14 days. The measured parameters included attachment rates, mortality rates, feeding duration, engorgement weight, oviposition rate, and egg weight.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.9. Statistics\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll data were expressed as mean ± SEM. Statistical significance was determined by Student’s t-test using SAS software (SAS Institute, 2013).\u0026nbsp;\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1. Codon optimization and cloning\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to ProteoGenix results (Additional File 1), CAI (Codon Adaptation Index) was estimated to be 0.68. Replacement of rare and less preferential codons with more favorable codon usage improved the expression level in E. coli. CAI was measured again to be 0.98 after codon optimization, which was within the optimum range.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe comparison of GC content between the original sequence and the optimized sequence of the voraxin\u0026nbsp;\u0026alpha;\u0026nbsp;is presented in Additional File 1. The Average GC content had a suitable value after (0.49) and before (0.58) optimizing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe codon-optimized of the voraxin\u0026nbsp;\u0026alpha;\u0026nbsp;sequence was synthesized and sub-cloned into pQE-30 by ProteoGenix Company. In recognition of sub-cloning, recombinant pQE30 was digested by Bam HI and Hind III. Accuracy of sub-cloning was confirmed by sequencing\u0026nbsp;(Additional File 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe ordered pQE-30 plasmid was transformed into competent E. coli cells\u0026nbsp;and sequenced for final confirmation after plasmid extraction (Additional File 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2. In vitro protein expression and western blot analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Fig. 1, the r-voraxin protein made in the pQE30 vector (considering the presence of 6x His tag on the N-terminus) was expressed as an 18 kDa protein on a 12% polyacrylamide gel, consistent with the predicted molecular mass.\u003c/p\u003e\n\u003cp\u003eNi-IDA Purification System was used to purify the recombinant protein carrying a 6x His tag. After the purification, the r-voraxin \u0026alpha; protein was solubilized in PBS, and used as antigen for western blot analysis. Western blot analysis was performed on protein extracts to detect the r-voraxin \u0026alpha; protein weight and their properties. The r-voraxin \u0026alpha; protein appeared as a single band with a molecular weight of 18 kDa (Fig. 2). These results indicated that the r-voraxin \u0026alpha; protein was synthetized in E.coli BL21 (DE3) cells in vitro. The concentration of proteins was estimated to be about 3.1 mg.mL-1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3. Serum antibody titers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe humoral response, three times at two-week intervals (0, 2, and 4 weeks), was detected by ELISA on serum from immunized and control rabbits before the tick challenge. As shown in Fig. 3, the IgG titer on the serum of the immunized rabbits significantly increased compared to the control, reaching 0.284 and 0.465 (mean value of three rabbits) in the second and fourth weeks, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4. Vaccination trials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeven, fourteen, and twenty-one days after the final immunization, sera obtained from immunized rabbits reacted with the r-voraxin \u0026alpha; antigen by Western blot analysis during the tick challenge (Fig 4). Western blot results confirmed the presence of an 18 kDa band for the r-voraxin \u0026alpha; antigen. As shown in Fig. 4, the r-voraxin \u0026alpha; antigen decreased over a 21-day time course.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5. Effects of the r-voraxin\u0026nbsp;\u003c/strong\u003e\u0026alpha;\u003cstrong\u003e\u0026nbsp;vaccine on tick feeding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe biological parameters included attachment rates, mortality rates, feeding duration, engorgement weight, oviposition rate, and egg weight measured to assess the immunogenic effect of the r-voraxin\u0026nbsp;\u0026alpha;\u0026nbsp;on female ticks during 14 days (Table 1). There was no observed difference between the vaccinated groups (84.0%) in comparison with the control group (85.0%) (Measured on day 5).\u0026nbsp;Also, there was no apparent difference in the feeding duration between vaccinated and control rabbits. The mortality rate in the vaccinated group increased compared to the control group (P \u0026lt; 0.01). In brief, 18/30 female ticks (60% of ticks fed on vaccinated rabbits) and 5/30 female ticks (16.6% of ticks fed on control rabbits) died during the vaccination trial.\u003c/p\u003e\n\u003cp\u003eAccording to the data presented in Table 1,\u0026nbsp;the weight of eggs and the body weight of engorged female ticks fed on vaccinated animals was significantly reduced compared to the control group (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.01).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAlso, the oviposition rate parameter was significantly decreased by 41.6% (5/12 ticks) in female ticks fed on vaccinated rabbits. While almost all live ticks (24/25 ticks) isolated from the control rabbits group laid live eggs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 1. Effects of the r-voraxin vaccine \u0026alpha; on \u003cem\u003eH. anatolicum\u003c/em\u003e Tick feeding\u003c/p\u003e\n\u003cdiv align=\"Left\"\u003e\n \u003ctable dir=\"rtl\" border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"600\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"51.919866444073456%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp dir=\"LTR\"\u003eImmunized group\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"48.080133555926544%\" valign=\"top\"\u003e\n \u003cp dir=\"LTR\"\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.166666666666668%\" valign=\"top\"\u003e\n \u003cp dir=\"LTR\"\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.833333333333332%\" valign=\"top\"\u003e\n \u003cp dir=\"LTR\"\u003eVaccinated\u0026nbsp;(r-voraxin)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"48%\" valign=\"top\"\u003e\n \u003cp dir=\"LTR\"\u003e\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.166666666666668%\"\u003e\n \u003cp dir=\"LTR\"\u003e85.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.833333333333332%\"\u003e\n \u003cp dir=\"LTR\"\u003e84.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"48%\" valign=\"top\"\u003e\n \u003cp dir=\"LTR\"\u003eAttachment rates (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.166666666666668%\"\u003e\n \u003cp dir=\"LTR\"\u003e8.5 \u0026plusmn; 0.75 (25)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.833333333333332%\"\u003e\n \u003cp dir=\"LTR\"\u003e7.5 \u0026plusmn;0.40 (12)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"48%\" valign=\"top\"\u003e\n \u003cp dir=\"LTR\"\u003eFeeding duration (days) of female tick (n)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.166666666666668%\"\u003e\n \u003cp dir=\"LTR\"\u003e\u0026nbsp;613.0 \u0026nbsp; \u0026nbsp; \u0026plusmn; 23.1 (25) \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.833333333333332%\"\u003e\n \u003cp dir=\"LTR\"\u003e159.0 \u0026plusmn; 25.7 ** (12)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"48%\" valign=\"top\"\u003e\n \u003cp dir=\"LTR\"\u003eFed weight (mg) of female tick (n)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.166666666666668%\"\u003e\n \u003cp dir=\"LTR\"\u003e295.4 \u0026plusmn; 9.5 \u0026nbsp;(25)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.833333333333332%\"\u003e\n \u003cp dir=\"LTR\"\u003e54.4 \u0026plusmn; 5.6 \u0026nbsp; ** (12)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"48%\" valign=\"top\"\u003e\n \u003cp dir=\"LTR\"\u003eWeight (mg) of egg mass (n)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.166666666666668%\"\u003e\n \u003cp dir=\"LTR\"\u003e16.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.833333333333332%\"\u003e\n \u003cp dir=\"LTR\"\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"48%\" valign=\"top\"\u003e\n \u003cp dir=\"LTR\"\u003eMortality rate\u003csup\u003eb\u003c/sup\u003e (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"23.166666666666668%\"\u003e\n \u003cp dir=\"LTR\"\u003e96\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.833333333333332%\"\u003e\n \u003cp dir=\"LTR\"\u003e58.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"48%\" valign=\"top\"\u003e\n \u003cp dir=\"LTR\"\u003eOviposition rate (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003ea)\u0026nbsp;Each group contains three rabbits. Six pairs of adult ticks were introduced into each ear bag (right and left). Results are expressed as the mean \u0026plusmn; SEM for each ear bag.\u003c/p\u003e\n\u003cp\u003eb)\u0026nbsp;Mortality rate was obtained from the ticks that could not survive during feeding.\u003c/p\u003e\n\u003cp\u003ec)\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.01 compared to the control (two-tailed Student\u0026rsquo;s t-test)\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eAlthough \u003cem\u003eH. anatolicum\u003c/em\u003e is recognized as one of the main infesting ticks in important livestock breeding areas, more effective anti-tick vaccines for \u003cem\u003eH. anatolicum\u003c/em\u003e are not yet available. Therefore, more studies are needed to identify candidate antigens for developing anti-tick vaccines against \u003cem\u003eH. anatolicum\u003c/em\u003e. Previous studies have shown that male tick-derived voraxinα from \u003cem\u003eAmblyomma hebraeum\u003c/em\u003e tick is essential for female engorgement and oviposition (Weiss and Kaufman, 2004). The biological parameters included the oviposition rate, egg weight, and body weight of female ticks decreased in animals vaccinated with r-voraxin α of \u003cem\u003eRhipicephalus appendiculatus\u003c/em\u003e (Yamada et al. 2009). These results have shown the possibility of r-voraxin α as a functional anti-tick antigen in \u003cem\u003eRhipicephalus appendiculatus\u003c/em\u003e. Recently, molecular cloning, characterization, and homologs of voraxin α were reported in \u003cem\u003eH. anatolicum\u003c/em\u003e (Nazari et al. 2022). In this report, the expression of the voraxin α protein was evaluated in the bacterial expression system as well as the immunogenicity effect of the r-voraxin α antigen in \u003cem\u003eH. anatolicum\u003c/em\u003e infestation as an anti-tick vaccine.\u003c/p\u003e \u003cp\u003eSince rare codons affect the expression level of recombinant proteins in the bacterial expression system, therefore codon optimization with preferred codons can increase the expression level in the host (Aghaei et al. 2021) and facilitate protein folding (Dali et al. 2011). For this purpose, the bacterial system was used for protein expression, and the codons of voraxin α were optimized. According to the results, the CAI showed a suitable and significant range for high expression after optimizing. CAI was measured to be 0.98 after codon optimization, which was within the optimum range. A CAI of 1.0 is considered perfect, while a CAI greater than 0.8 is rated suitable for expression in the desired expression organism. Therefore, there is a greater chance of protein expression at a high level (Carbone 2003). Therefore, the efficiency of voraxin α production increased by using an optimized codon. The Average GC content had a suitable value after optimization. The ideal percentage range of GC content is between 30 and 70%. The values outside of this range will affect the efficiency of transcription (Puigbo 2007).\u003c/p\u003e \u003cp\u003eThe recombinant protein was purified by Ni2\u0026thinsp;+\u0026thinsp;affinity chromatography and then confirmed by SDS-PAGE and western blot analysis. Recently, reports indicated that these tags do not affect the efficiency of r-voraxin α. In addition, similar results were observed regarding the molecular weight (18 kDa) of r-voraxinα expressed in E. coli Yamada et al. 2009; Weiss and Kaufman, 2004).\u003c/p\u003e \u003cp\u003eIn the vaccination trial, r-voraxin α induced humoral immune response in immunized rabbits, as determined by ELISA, suggesting that rabbit antibodies elicited upon vaccination could react with the r-voraxin α protein.\u003c/p\u003e \u003cp\u003eConsistent with this research, previous studies indicated that the immunization trial increased the mortality rate and reduced the egg weight, oviposition rate, and engorgement weight of female ticks upon challenge infestation of \u003cem\u003eH. anatolicum\u003c/em\u003e in rabbits (Yamada et al. 2009; Weiss and Kaufman, 2004). The mortality rate obtained in this research (60%) was higher than the mortality rate reported by Yamada (26.7%) (Yamada et al. 2009). Reduction in the egg weight and engorgement weight of female ticks showed in this study were approximately 75% and 81%, whereas the egg weight and engorgement weight of female ticks reported by Yamada et al. (2009) were 50% and 40%, respectively. Weiss and Kaufman (2004) reported that the mean weight of females fed on immunized rabbits showed a 72% reduction compared with the normal females fed on control rabbits infected with \u003cem\u003eAmblyomma hebraeum\u003c/em\u003e tick. In all previous reports, the voraxin α was expressed without codon optimization. The \u003cem\u003eAmblyomma hebraeum\u003c/em\u003e voraxin α was expressed in the Sf21 insect cell line (Weiss and Kaufman 2004). Also, the \u003cem\u003eR. appendiculatus\u003c/em\u003e voraxin α expression was performed in E. coli strain Rosetta-gami DES LysS and \u003cem\u003eDrosophila\u003c/em\u003e Schneider 2 (S2) insect cells without codon optimization. Although the expression occurred in both bacterial and insect cells, the results showed that the bacterial cell is more suitable (Yamada et al. 2009). The reason for the greater effectiveness of voraxin α may be related to the optimization of the voraxin α codon in this study. Codon optimization enhances protein expression and folding in the host.\u003c/p\u003e \u003cp\u003eEven though there were no differences in the attachment rates between vaccinated and control groups, an apparent increase in the mortality rate indicated that vaccination of rabbits with the r-voraxin α had a protective effect against \u003cem\u003eH. anatolicum\u003c/em\u003e ticks.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eCodons of \u003cem\u003eH. anatolicum\u003c/em\u003e voraxin α sequence were optimized according to the codon usage in E. coli. A codon-optimized voraxin α was synthesized into a pQE30 expression vector. The r-voraxin α expression were successfully performed in E. coli. The r-voraxin α protein was purified from cellular extraction and appeared as a single band with a molecular weight of 18 kDa. The results of this research suggested that the codon-optimized \u003cem\u003eH. anatolicum\u003c/em\u003e voraxin α sequence expressed in the bacterial expression system could be considered a vaccine with significant protective potential for immunized hosts against \u003cem\u003eH. anatolicum\u003c/em\u003e tick infestation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe\u0026nbsp;authors\u0026nbsp;confirm that there are no known conflicts of interest\u0026nbsp;associated with this publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors confirm that the data supporting the findings of this study are available within the article (and/or) its supplementary materials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe hereby declare, all ethical standards have been respected in preparation of the submitted article.\u003c/p\u003e\n\u003cp\u003eAuthor Contribution\u003c/p\u003e\n\u003cp\u003e\u0026quot;M.N conceived and designed the experiments. Z.M collected ticks and conducted experiments in farms and labs with helping M.N and M.R.T. Also, H.R and F.S analyzed the data. F.S and M.N wrote the manuscript. All authors have read and approved the manuscript\u0026quot;.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAghaei SH, Saffar B, Ghaedi K, Mobini-Dehkordi M. Functional analysis of recombinant codon-optimized bovine neutrophil \u0026beta;-defensin. J Adv Res 2022; 38: 299. https://doi.org/10.1016/j.jare.2021.06.002\u003c/li\u003e\n \u003cli\u003eAlmazan C, Lagunes R, Villar M, Canales M, Rosario-Cruz R, Jongejan F, de la Fuente J. Identification and characterization of Rhipicephalus (Boophilus) microplus candidate protective antigens for the control of cattle tick infestations. 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Proc Natl Acad Sci USA 1989; 86: 9657\u0026ndash;9661. https://doi:10.1073/pnas.86.24.9657\u003c/li\u003e\n \u003cli\u003eSambrook J, Green MR. Molecular Cloning, A Laboratory Manual, 4th Edition, Cold Spring Harbor Protocols, New York, 2012. www.cshprotocols.org.\u003c/li\u003e\n \u003cli\u003eSAS Institute. (2013). SAS Users Guide: Statistics. Version 9.4. SAS Institute Inc., Cary, NC.\u003c/li\u003e\n \u003cli\u003eSonenshine DE. Evolution and systematic relationships of ticks, (chapter 2) In: Biology of Ticks, Oxford University Press, New York. 1991; 1: 13-50\u003c/li\u003e\n \u003cli\u003eWaladde SM, Ochieng SA, Gichuhi PM. Artificial membrane feeding of the ixodid tick, Rhipicephalus appendiculatus, to repletion. Experimental and applied acarology 1991; 11 (4): 297-396. https://doi.org/10.1007/BF01202876\u003c/li\u003e\n \u003cli\u003eWeiss BL, Kaufman WR. Two feeding-induced proteins from the male gonad trigger engorgement of the female tick, Amblyomma hebraeum. Proc Natl Acad Sci USA 2004; 101(16): 5874\u0026ndash;9. https://doi:10.1073/pnas.0307529101.\u003c/li\u003e\n \u003cli\u003eYamada S, Konnai S, Imamura S, Ito T, Onuma M, Ohashi K. Cloning and characterization of Rhipicephalus appendiculatus voraxin and its effect as anti-tick vaccine. Vaccine 2009; 27(43): 5989\u0026ndash;5997. https://doi: 10.1016/j.vaccine.2009.07.072.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Hyalomma anatolicum, Ticks, Voraxin α, vaccine, Codon optimization","lastPublishedDoi":"10.21203/rs.3.rs-3865639/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3865639/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eResearch has shown that voraxin α derived from male ticks stimulates blood feeding to engorge in female ticks. Whereas, the oviposition rate, egg weight, and body weight of female ticks were reduced in animals vaccinated with recombinant (r-) voraxin α. These data suggest a potential role of r-voraxin α as a functional anti-tick antigen in \u003cem\u003eRhipicephalus\u003c/em\u003e \u003cem\u003eappendiculatus \u003c/em\u003eand \u003cem\u003eAmblyomma\u003c/em\u003e \u003cem\u003ehebraeum\u003c/em\u003e tick infestation. This study investigated the immunogenicity of r-voraxin α protein from \u003cem\u003eHyalomma anatolicum\u003c/em\u003e (\u003cem\u003eH. anatolicum\u003c/em\u003e) tick as an anti-tick vaccine in rabbits. The \u003cem\u003eH. anatolicum\u003c/em\u003e voraxin α sequence was optimized according to the codon usage in E. coli before being sub-cloned into pQE30. The gene sequence of the voraxin α was synthesized, verified by DNA sequencing, cloned in a pQE30 vector, and transformed into E. coli. Then, the expression of the r-voraxin α protein was confirmed by SDS-PAGE and Western blot analysis. Subsequently, three rabbits were immunized with the r-voraxin α as the vaccinated group, whereas three rabbits without injection were considered the control group. The result indicated the success of cloning of codon-optimized \u003cem\u003eH. anatolicum\u003c/em\u003e voraxin α gene. Moreover, the expression of the r-voraxin α protein (approximately 18 kDa) in the bacterial expression system was confirmed by SDS-PAGE and Western blot analysis. The results of this study showed that the mortality rate in vaccine recipients increased compared to the control group (\u003cem\u003eP \u0026lt; 0.01\u003c/em\u003e). Also, the egg weight, oviposition rate, and engorgement weight of female ticks fed from vaccinated animals were significantly reduced compared to the control group (\u003cem\u003eP \u0026lt; 0.01\u003c/em\u003e). The results confirmed that the codon-optimized \u003cem\u003eH. anatolicum\u003c/em\u003evoraxin α gene expressed in the bacterial expression system could be a suitable anti-tick vaccine against \u003cem\u003eH. anatolicum\u003c/em\u003e tick infestation.\u003c/p\u003e","manuscriptTitle":"Codon optimization of voraxin α sequence enhances the immunogenicity of a recombinant vaccine against Hyalomma anatolicum infestation in rabbits","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-18 09:16:30","doi":"10.21203/rs.3.rs-3865639/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c68458b7-73f3-42ef-bd2f-9d4136bb20e8","owner":[],"postedDate":"January 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-05-08T10:23:41+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-18 09:16:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3865639","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3865639","identity":"rs-3865639","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}