Experimental and Molecular Predictions of the Adjuvanticity Of

bioRxiv preprint doi: https://doi.org/10.1101/2021.01.29.428769; this version posted January 29, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 2 3 4 5 Experimental and Molecular Predictions of the 6 Adjuvanticity of Snail Mucin on Hepatitis B Vaccine in 7 Albino Mice 8 9 10 Parker E. Joshua 1¶, Cynthia O. Nwauzor 1¶, Damian C. Odimegwu2&, Uzochukwu G. 11 Ukachukwu 1&, Rita O. Asomadu1& and Timothy P. C. Ezeorba 1¶*, 12 13 1Department of Biochemistry, Faculty of Biological Sciences, University of Nigeria, Nsukka 14 2Department of Pharmaceutical Microbiology & Biotechnology, University of Nigeria, Nsukka 15 16 *Corresponding Author’s Details: 17 Email: [email protected] (TPCE) 18 19 20 ¶ These authors contributed equally to this work. 21 &These authors also contributed equally to this work 22 23 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.29.428769; this version posted January 29, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 24 Abstract 25 Although aluminum-containing adjuvants are widely used in human vaccination due to 26 their excellent safety profile, they exhibit low effectiveness with many recombinant antigens. This 27 study investigated the adjuvanticity of snail mucin with recombinant Hepatitis B Vaccine 28 (rHBsAg). Twenty-five (25) female mice distributed unbiasedly into 5 groups were used in the 29 study and were administered different rHBsAg/Mucin formulation at 7 days intervals. Blood 30 samples were collected a day following the administration for analysis. The results of liver 31 function and body weight analysis were indications that snail mucin had no adverse effect on the 32 mice. The treatment group (administer mucin and rHBsAg) showed significantly (P<0.05) higher 33 mean titres of anti-HBsAg antibodies when compared with the negative controls and the positive 34 control administered with two doses of rHBsAg. Furthermore, a comparable immune response 35 to positive control administered with three doses rHBaAG was recorded. In silico prediction, 36 studies of the protein-protein interaction of a homology modelled snail mucus protein and HBsAg 37 gave an indication of enhanced HBV antigen-antibody interaction. Therefore, this study has 38 shown that snail mucin possesses some adjuvant properties and enhances immune response 39 towards rHBsAg vaccine. However, there is a need for further molecular dynamics studies to 40 understand its mechanism of action. 41 Keywords: Snail mucin, Adjuvants, recombinant antigen, immunogenicity, Homology 42 Modelling 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.29.428769; this version posted January 29, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 43 Introduction 44 Hepatitis B Virus (HBV) is a causative agent of hepatitis B infection, Approximately, a 45 million people die annually from HBV-related chronic liver diseases, such as liver failure, liver 46 cirrhosis and hepatocellular carcinoma (HCC) [1]. The disease is majorly transmitted across 47 human population through unprotected sex, birth transmission, transfusion of contaminated 48 blood, and the use of object that are contaminated [2]. 49 Vaccination is the most effective measure to decrease the worldwide HBV prevalence and 50 its complications [3]. Generally, vaccination is aimed to induce protective immunity against a 51 unique epitope of an antigen, and in some vaccines, this can be enhanced by addition of adjuvants. 52 Adjuvants are substances added to vaccines to improve the immune response of pure antigens, 53 which may not stimulate adequate immune response on their own [4] 54 For several decades, aluminium containing adjuvants (alums) have been very effective in 55 human vaccination and were generally the only approved adjuvants by the United States Food 56 and Drug Administration (FDA) due to its’ excellent track record of safety, low cost and its 57 application with variety of antigens. However, in the modern era of recombinant proteins and 58 small peptides vaccination, alums are implicated with a number of limitations such as local 59 reactions, zero effectiveness to some recombinant antigens, and poor augmentation ability to 60 some cell-mediated immune responses, such as cytotoxic T-cell responses [5]. Hence, there are 61 several ongoing studies to investigate other effective adjuvants such as biodegradable polymeric 62 particle technology. 63 The discovery of a safe and effective HBV vaccine derived from yeast-derived 64 recombinant hepatitis B surface antigen (rHBsAg) promises a global reduction in HBV incidence. 65 However, to generate the effective immune responses, individuals need about three or more doses 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.29.428769; this version posted January 29, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 66 of the vaccine after a couple of months interval [6]. Consequently, maintaining a consistent re- 67 immunization rate for multiple-administration program is quite difficult, especially in developing 68 countries [7]. Hence, this study is birthed from the need for the development of more effective 69 adjuvant/vaccine delivery systems against HBV, possibly requiring only a single round of 70 immunization to yield long-lasting immune responses. 71 Many recent studies on adjuvant development for hepatitis B vaccines are now focusing 72 on the use of biodegradable polymeric particles (BPP) for adjuvants and have reported promising 73 results [1]. Snail mucin being a natural mucoadhesive polymer can be classified as a BPP owing 74 to its biocompatibility, non-antigenic/non-toxic nature as well as biodegradability in the living 75 system [8]. Many recent studies have discovered snail mucin to possess wound healing and age 76 renewing properties [9]. Other studies further reported that snail mucin possesses diverse 77 biophysical and pharmaceutical applications and are effective for mucoadhesive drug delivery 78 agent [10]. In this study, the suitability of snail mucin polymer as an adjuvant for recombinant 79 HBsAg vaccine was investigated in albino mice. 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.29.428769; this version posted January 29, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 81 2. Materials and Methods 82 2.1 Reagents 83 All chemicals and reagents used for the study were of analytical grade and included 2 M 84 H2SO4 (JHD), 96% Acetone (JHD), Alanine aminotransferase (ALT) Kit (DiaLab, Austria), 85 Aspartate aminotransferase (AST) Kit (DiaLab, Austria), Carbonate-Bicarbonate Buffer (pH 9.5), 86 Citrate Phosphate Buffer (pH 5.0), DMSO (JHD, China), methanol, Fat-free Milk powder (Dano- 87 Slim, Nigeria), Hydrogen Peroxide (JHD, China), Phosphate Buffered Saline (pH 7.4), Leishman 88 stain, Tetramethylbenzidine (TMB) Tablets – (SIGMA, USA), Tween 20 (Germany). 89 2.2 Vaccine and Secondary Antibodies 90 Recombinant hepatitis B surface antigen vaccine, Euvax-B (LG Chemical, South Korea) 91 was used as the vaccine candidate for immunization. Goat anti-mouse horseradish peroxidase- 92 conjugated IgG, IgG1 and IgG2a immunoglobulins (Southern Biotechnology, USA) were used 93 as secondary antibodies. Both vaccine and secondary antibodies were stored at 4oC until needed. 94 2.3 Snails 95 Adult giant African land snails (Archachatina marginata) were procured from the Orie- 96 Orba market, Enugu, Nigeria. A total number of 130 snails were used and were identified in the 97 Department of Zoology and Environmental Biology, University of Nigeria. 98 2.4 Animals 99 Female albino mice aged 6 to 8 weeks were used for the study. The animals were obtained 100 from the Animal house of the Faculty of Veterinary Medicine, University of Nigeria and were 101 used for the experimental study. They were acclimatized for 10 days under optimised 5 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.29.428769; this version posted January 29, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 102 environmental conditions with a 12-hour light/dark cycle and fed constantly with tap water and 103 standard livestock feeds from Pfizer Livestock Feeds Plc, Nigeria. 104 2.5 Extraction of Snail Mucin 105 The study adopted the method published by Adikwu and Ikejiuba [11] to extract the mucin 106 from the giant African land snail. 107 2.6 Preparation of Snail Mucin Dispersion (Adjuvant) 108 Snail mucin adjuvant was prepared according to the method of Adikwu and Nnamani [12]. 109 A quantity

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