Experimental and molecular predictions of the adjuvanticity of snail mucin on hepatitis B vaccine in albino mice

Although aluminum-containing adjuvants are widely used in human vaccination due to their excellent safety profile, they exhibit low effectiveness with many recombinant antigens. This study investigated the adjuvanticity of snail mucin with recombinant Hepatitis B Vaccine (rHBsAg). Twenty-five (25) female mice distributed unbiasedly into 5 groups were used in the study and were administered different rHBsAg/Mucin formulation at 7 days intervals. Blood samples were collected a day following the administration for analysis. The results of liver function and body weight analysis were indications that snail mucin had no adverse effect on the mice. The treatment group (administer mucin and rHBsAg) showed significantly (P<0.05) higher mean titres of anti-HBsAg antibodies when compared with the negative controls and the positive control administered with two doses of rHBsAg after the boost doses (day 28). Furthermore, a comparable immune response to positive control administered with three doses rHBaAG was recorded. In silico prediction, studies of the protein-protein interaction of a homology modelled snail mucus protein and HBsAg gave an indication of enhanced HBV antigen-antibody interaction. Therefore, this study has shown that snail mucin possesses some adjuvant properties and enhances immune response towards rHBsAg vaccine. However, there is a need for further molecular dynamics studies to understand its mechanism of action.


Determination of Body Weights and Liver weight
The mice were weighed daily throughout the study duration (from day 0 to day 28) using 132 an electronic weighing scale. On day 28, the mice were sacrificed, and their livers were harvested 133 and weighed using an electronic weighing scale. Liver weights were recorded. The mice differential white blood cell count was determined on days 0, 14, 21 and 28 136 using the method described by Carr and Rodak [13].  138 Vaccine. 139 Serum AST and ALT concentrations were determined using DiaLab diagnostic kits 140 (Wiener Neudorf, Austria). Determination of AST and ALT activities were based on the method 141 described by Thomas [14], Moss & Henderson [15]. An abnormal increase in the amount of AST 142 and ALT in the blood above the normal threshold is an indication of liver disease or damaged 143 hepatic cells. Principally, serum Aspartate Aminotransferase (AST) and Serum Alanine 8 144 Aminotransferase (ALT) activity were determined quantitatively by its enzymatic effect on the 145 oxidation of NADH to NAD + . 146 2.12 Measurement of Antibody Titers of IgG, IgG1 and IgG2a Against rHBsAg Using 147 Indirect Enzyme-linked Immunosorbent Assay (iELISA) 148 Analysis of specific total antibodies (IgG) were performed using an optimized indirect 149 ELISA method described by Nejati et al. [18] on a 96-well ELISA plates (BRANDplates®,150 Germany) coated with 100 µL of rHBsAg. Similarly, the specific IgG1 and IgG2a subclasses 151 were performed using goat anti-mouse IgG1 and IgG2a secondary antibodies (Southern 152 biotechnology, USA).

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The results were expressed as mean ± standard deviation (S.D). Data were analyzed using 155 SPSS using one-way and two-way ANOVA and subjected to Duncan tests. Differences between 156 the mean of the treated groups and control would be considered significant at P < 0.05.

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The modelled HBsAg 3D structure was docked to both the immunoglobulin structure  Table 1, the AST activity of the 2DVacSM experimental group was non-12 220 significantly (p>0.05) higher when compared to other experimental groups and the controls.

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Moreover, the value was within the normal range of AST for mice (54-298 U/L).

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Similarly, the ALT activity of the 2DVacSM and 2DSM experimental group fell with the 223 normal range of ALT enzyme activities for mice (17-77 U/L) and showed non-significantly 224 (p>0.05) difference when compared to other groups and the control (Table 1).  To observe the effect of Snail Mucin-Adjuvanted rHBsAg Vaccine on lymphocytes, a 245 differential white blood cell count was performed. There was a significant (p<0.05) decrease in 246 Lymphocyte count for all experiment groups from day 0 to day 28 (Fig. 3).

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Performing a sequence blast of snail mucus protein against the protein data bank, the best 273 structural homolog being the structure of proximal thread matrix protein 1 (PTMP1) from the 274 mussel byssus, was selected to generate the homologous model of the snail mucus protein.

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The homology modelled snail mucus structure (Fig. 5a)  predictions from the docking experiment, the morph, gave a lower negative score of -2646.06.

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Meanwhile, the best prediction from docking the standalone HBsAg gave a higher negative score 309 of -4296.27. Although the score from the Morph docking was not better than the standalone 310 antigen, it was discovered that both the Morph and the crystal Pres1 antigen interacted with the 311 similar variable region for the Fab Fragment of the antibody (Fig. 7), while the best prediction of 312 the full HBsAg did not bind to the either variable region of the Fab fragments. immune responses. They are highly important in phagocytosis, antigen presentation, cytokine 389 production, and antimicrobial and cytotoxic activities [28]. Thus, stimulation of monocytes is an 390 indication that snail-mucin adjuvanted rHBsAg vaccine is more immunogenic than the vaccine 391 alone.

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No significant basophil release was detected in any of the groups. This is in line with

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O'Connell et al. [29], who reported that basophils are rarely present in murine peripheral blood.

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The absence of basophils in the group co-administered snail mucin and vaccine proves that the 395 vaccine formulation did not produce any allergic reaction.

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There was an upregulation of eosinophils in the group co-administered snail mucin and Co-administration of snail mucin with vaccine caused significant increases in neutrophil 413 counts compared to administration of vaccine alone (Fig. 2). There was a corresponding decrease 414 in lymphocyte count. This is in consonance with Provencher et al. [34] who stated that in mice,