In Silico Analysis of Ixodid Tick Aqauporin-1 Protein as a Candidate Anti-Tick Vaccine Antigen

Ticks are arthropod vectors of pathogens of both Veterinary and Public health importance. Ticks are largely controlled by acaricide application. However, acaricide efficacy is hampered by high cost, the need for regular application and selection of multi-acaricide resistant tick populations. In light of this, future tick control approaches are poised to rely on integration of rational acaricide application and other methods such as vaccination. To contribute to systematic research-guided efforts to produce anti-tick vaccines, we carried out an in silico tick Aquaporin-1 protein (AQP1) analysis to identify unique tick AQP1 peptide motifs that can be used in future peptide anti-tick vaccine development. We used multiple sequence alignment (MSA), motif analysis, homology modeling, and structural analysis to identify unique tick AQP1 peptide motifs. BepiPred, Chou & Fasman-Turn, Karplus & Schulz Flexibility and Parker-Hydrophilicity prediction models were used to asses these motifs’ abilities to induce antibody mediated immune responses. Tick AQP1 (MK334178) protein homology was largely similar to the bovine AQP1 (PDB:1J4N) (23% sequence similarity; Structural superimposition RMS=1.475). The highest similarities were observed in the transmembrane domains while differences were observed in the extra and intra cellular protein loops. Two unique tick AQP1 (MK334178) motifs, M7 (residues 106-125, p=5.4e-25) and M8 (residues 85-104, p=3.3e-24) were identified. These two motifs are located on the extra-cellular AQP1 domain and showed the highest Parker-Hydrophilicity prediction immunogenic scores of 1.153 and 2.612 respectively. The M7 and M8 motifs are a good starting point for the development of potential peptide-based anti-tick vaccine. Further analyses such as in vivo immunization assays are required to validate these findings.

4 66 are associated with selection of multi-acaricide resistant tick populations [10]; a problem that has 67 been on the rise in Uganda [11]. In light of this, future tick control strategies will have to depend 68 on integration of economically effective acaricide application, vaccination, breeding livestock for 69 tick resistance, and other available tick control methods such as controlled animal movements [9].
70 To date anti-tick vaccine development has been slow, for example the only BM86 recombinant 71 vaccine against R. microplus was developed more than 2 decades ago [12]. There is therefore a 72 need to rekindle systematic research-guided efforts to evaluate crucial tick proteins and biological 73 pathways for vaccine development.

74
Tick aquaporins belong to the Membrane Intrinsic Protein (MIP) superfamily which are 75 known to play a key role in transport of water and glycerol across the cell membrane. MIP has 76 three subfamilies; Classical aquaporin (cAQP), aquaglyceroporin and S-aquaporin. The major 77 difference between the three subfamilies is in the signature residues around the protein pore.
78 Classical aquaporin allows only the passage of water, while aquaglyceroporin take in glycerol. In 79 ticks, there are two types of aquaporins namely: AQP1 and AQP2 which help in concentrating 80 blood meals by facilitating the excretion of excess water back into the host via the salivary glands 81 [13,14]. From mRNA analysis of female I. Ricinusi ticks, it was shown that AQP1 is expressed in 82 tissues such as the gut, rectal sac and most abundantly in the salivary glands where it is involved 83 in water flux, while AQP2 is only expressed in the salivary glands [ 251 Phylogenetic analysis further confirmed this divergence where tick AQP1 sequences grouped in a 252 distinct clade while human and bovine AQP1 clustered together in a separate clade (Fig 5). The amino acid sequences were additionally analysed using Pearson correlation matrix that 263 revealed that tick AQP1 had a within positive correlation and a negative correlation between 264 bovine and human AQP1 amino acid sequences ranging -0.6 to -0.4 (Fig 6). These results imply 13 265 that much as AQP1 is conserved among different species, some differences exist between tick 266 AQP1 and bovine or human AQP1 proteins at the sequence level.  (Fig 7). The NPA is a highly conserved hydrophobic motif that forms part of the pore 279 for each AQP1 monomer [36]. In addition, tick AQP1 motifs contained an aspartic acid (D) residue 280 after the second NPA motif, a signature for aquaglyceroporins (Fig 7). Motif analysis using MEME revealed six AQP1 motifs located on the extra and intra 289 cellular domains of the homologous tick AQP1 that were unique to ticks (Fig 8). These motifs 290 included; M3 (residues 2-11), M7 (residues 12-31), M8 (residues 85-104), M9 (residues 106-125), 291 M10 (residues 149-168), and M16 (residues 193-212) (Fig 8). 306 respectively) which are colored in black (Fig 9). The most highly conserved motif was M16 with 307 over 70% of its residues being hydrophobic with most of the motif located in the transmembrane 308 domain. Motif M7 contained a high number of substitutions and all these alterations were observed 309 in one tick species (I. sanguine sequences XP002399532) with a p value of 5.4e-25. Motifs M7 310 and M8 contained mostly hydrophilic residues (Fig 9). 319 Homology model04 (Fig 10-A and 10-B) that had the lowest z-DOPE (normalised Discrete 320 Optimized Protein Energy) score of -0.580 (Table 1) and a ProSA z-score of -3.72 was selected 321 for use in further analyses (Fig 10-C). A z-DOPE score close to -1 indicates that the protein model 322 is close to the native structure [37]. Structure super-imposition of the model proteins and the 323 templates further confirm that model04 is more similar to templates protein (1FX8 and 1LDF) 324 with a Root Mean Square Deviation (RMSD) of 0.34 and 0.36 respectively. A Ramachandran plot 325 score analysis of these models further revealed that model04 had 170 (89.9%) of its residues in the 326 most favoured regions, 16 (8.5%) residues in the additional allowed region and 3 (1.6%) residues 327 in generously allowed region. (Fig 10-D). The NPA1, NPA2 and ar/R filter are shown in Fig 10-328 A while Fig 10-B shows residues of the amino acid residues of the ar/R filter 329  The six motifs were then mapped onto the modeled AQP1 protein structure (model04) to 343 determine their location on the protein 3D structure. Motifs M9, M10, and M16 were localised on 344 the transmembrane protein domain, while motifs M3, M7 and M8 on the extracellular protein 345 domain (Fig 11). The structural superimposition of the tick AQP1 homology model04 (Fig 12-A In this study, in silico-based methods were used to predict AQP1 peptide motifs unique to 370 ticks isolated from this study and other AQP1 from the Protein Data Bank. The predicted motifs 371 were further assessed for their potential of inducing humoral immune response and therefore their 372 potential for being good vaccine antigens. We identified a total of 6 unique tick-AQP1 peptide 373 motifs.

374
Tick AQP1 proteins from this study shown regions of similarity to AQP1 from other tick 375 species and other organisms (Fig 7). This is due to the obvious fact that AQP1 plays similar