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

Parker Elijah Joshua; Cynthia Ogochukwu Nwauzor; Damian Chukwu Odimegwu; Uzochukwu Gospel Ukachukwu; Rita Onyekachukwu Asomadu; Timothy Prince Chidike Ezeorba

doi:10.1371/journal.pone.0246915

. 2021 Jul 23;16(7):e0246915. doi: 10.1371/journal.pone.0246915

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

Parker Elijah Joshua ^1,^#, Cynthia Ogochukwu Nwauzor ^1,^#, Damian Chukwu Odimegwu ^2,^‡, Uzochukwu Gospel Ukachukwu ^1,^‡, Rita Onyekachukwu Asomadu ^1,^‡, Timothy Prince Chidike Ezeorba ^1,^*,^#

Editor: Isabelle Chemin³

PMCID: PMC8301616 PMID: 34297725

Abstract

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.

Introduction

Hepatitis B Virus (HBV) is a causative agent of hepatitis B infection, Approximately, a million people die annually from HBV-related chronic liver diseases, such as liver failure, liver cirrhosis and hepatocellular carcinoma (HCC) [1]. The disease is majorly transmitted across human population through unprotected sex, birth transmission, transfusion of contaminated blood, and the use of object that are contaminated [2].

Vaccination is the most effective measure to decrease the worldwide HBV prevalence and its complications [3]. Generally, vaccination is aimed to induce protective immunity against a unique epitope of an antigen, and in some vaccines, this can be enhanced by addition of adjuvants. Adjuvants are substances added to vaccines to improve the immune response of pure antigens, which may not stimulate adequate immune response on their own [4].

For several decades, aluminium containing adjuvants (alums) have been very effective in human vaccination and were generally the only approved adjuvants by the United States Food and Drug Administration (FDA) due to its’ excellent track record of safety, low cost and its application with variety of antigens. However, in the modern era of recombinant proteins and small peptides vaccination, alums are implicated with a number of limitations such as local reactions, zero effectiveness to some recombinant antigens, and poor augmentation ability to some cell-mediated immune responses, such as cytotoxic T-cell responses [5]. Hence, there are several ongoing studies to investigate other effective adjuvants such as biodegradable polymeric particle technology.

The discovery of a safe and effective HBV vaccine derived from yeast-derived recombinant hepatitis B surface antigen (rHBsAg) promises a global reduction in HBV incidence. However, to generate the effective immune responses, individuals need about three or more doses of the vaccine after a couple of months interval [6]. Consequently, maintaining a consistent re-immunization rate for multiple-administration program is quite difficult, especially in developing countries [7]. Hence, this study is birthed from the need for the development of more effective adjuvant/vaccine delivery systems against HBV, possibly requiring only a single round of immunization to yield long-lasting immune responses.

Many recent studies on adjuvant development for hepatitis B vaccines are now focusing on the use of biodegradable polymeric particles (BPP) for adjuvants and have reported promising results [1]. Snail mucin being a natural mucoadhesive polymer can be classified as a BPP owing to its biocompatibility, non-antigenic/non-toxic nature as well as biodegradability in the living system [8]. Many recent studies have discovered snail mucin to possess wound healing and age renewing properties [9]. Other studies further reported that snail mucin possesses diverse biophysical and pharmaceutical applications and are effective for mucoadhesive drug delivery agent [10]. In this study, the suitability of snail mucin polymer as an adjuvant for recombinant HBsAg vaccine was investigated in albino mice.

Materials and methods

Animals

The Faculty of Biological Sciences, University of Nigeria, Board on Ethical Clearance for Animal Research granted approval to perform the research on Albino mice. Female albino mice aged 6 to 8 weeks were used for the study. The animals were obtained from the Animal house of the Faculty of Veterinary Medicine, University of Nigeria and were used for the experimental study. They were acclimatized for 10 days under standard environmental conditions with a 12-hour light/dark cycle, fed with standard pellets (Pfizer Livestock feeds Plc, Enugu, Nigeria) and tap water was given ad libitum. Precaution were taken to ensure that the blood collection procedure did not result to the loss of the animal sight, thereby affecting their feeding and other physiological activities.

Reagents

All chemicals and reagents used for the study were of analytical grade and included 2 M H₂SO₄ (JHD), 96% Acetone (JHD), Alanine aminotransferase (ALT) Kit (DiaLab, Austria), Aspartate aminotransferase (AST) Kit (DiaLab, Austria), Carbonate-Bicarbonate Buffer (pH 9.5), Citrate Phosphate Buffer (pH 5.0), DMSO (JHD, China), methanol, Fat-free Milk powder (Dano-Slim, Nigeria), Hydrogen Peroxide (JHD, China), Phosphate Buffered Saline (pH 7.4), Leishman stain, Tetramethylbenzidine (TMB) Tablets–(SIGMA, USA), Tween 20 (Germany).

Vaccine and secondary antibodies

Recombinant hepatitis B surface antigen vaccine, Euvax-B (LG Chemical, South Korea) was used as the vaccine candidate for immunization. Goat anti-mouse horseradish peroxidase-conjugated IgG, IgG1 and IgG2a immunoglobulins (Southern Biotechnology, USA) were used as secondary antibodies. Both vaccine and secondary antibodies were stored at 4°C until needed.

Snails

Adult giant African land snails (Archachatina marginata) were procured from the Orie-Orba market, Enugu, Nigeria. A total number of 130 snails were used and were identified in the Department of Zoology and Environmental Biology, University of Nigeria.

Extraction of snail mucin

The study adopted the method published by Kalu et al. [11] to extract the mucin from the giant African land snail.

Preparation of snail mucin dispersion (adjuvant)

Snail mucin adjuvant was prepared according to the method of Adikwu and Nnamani [12]. A quantity (0.01g) of powdered snail mucin extract was weighed using an electronic balance and dispersed in 1 ml of normal saline. The dispersion was stirred for 15 minutes and allowed to hydrate completely for 24 hours before use. Thus, the concentration of the snail mucin was 1% w/v.

Experimental design

A total of 25 animals were randomly divided into five (5) groups of five (5) animals each as follows:

Group 1 (3DVac): First positive control (rHBsAg vaccine alone: 3 doses)
Group 2 (2DVac): Second positive control (rHBsAg vaccine alone: 2 doses)
Group 3 (2DVacSM): First treatment group (rHBsAg vaccine + Snail mucin: 2 doses)
Group 4 (2DSM): Second treatment group (Snail mucin alone: 2 doses).
Group 5 (3NS): Negative control (Normal saline: 3 doses)

Immunization protocol

Euvax-B (LG-Chemical, South Korea) was employed as a vaccine candidate. The mice were injected intramuscularly in a prime-boosting fashion with either 100μL (2μg) of rHBsAg vaccine alone (3 and 2 doses respectively), 100 μL of rHBsAg vaccine + 50 μL of snail mucin, 50 μL of snail mucin alone, or 100 μL of normal saline. The animals were vaccinated on days 1 and 15 (for 2 doses) and on days 1, 15 and 22 (for 3 doses) by a well-trained researcher in animal handling. Extensive efforts were deployed to minimize animal suffering during does administration and animal handling.

An aliquot of 0.5 mL of blood samples were collected from all experimental mice on days 0, 14, 21 and 28 before immunizations through the retro-orbital plexus and centrifuged at 5000 rpm for 10 minutes to obtain clear sera which were frozen until needed for immunochemical analyses.

Determination of body weights and liver weight

The mice were weighed daily throughout the study duration (from day 0 to day 28) using an electronic weighing scale. On day 28, the mice were euthanized by the method of cervical dislocation and their livers were harvested and weighed using an electronic weighing scale. The protocol of euthanasia by cervical dislocation was performed quickly by trained researcher, to ensure painless animal sacrifice. No anaesthesia or analgesia administer to the animal, to prevent interference with other histopathology analysis.

Differential white blood cell count

The mice differential white blood cell count was determined on days 0, 14, 21 and 28 using the method described by Carr and Rodak [13].

Liver function test of mice immunized with snail mucin-adjuvanted rHBsAg vaccine

Serum AST and ALT concentrations were determined using DiaLab diagnostic kits (Wiener Neudorf, Austria). Determination of AST and ALT activities were based on the method described by Thomas [14], Moss & Henderson [15]. An abnormal increase in the amount of AST and ALT in the blood above the normal threshold is an indication of liver disease or damaged hepatic cells. Principally, serum Aspartate Aminotransferase (AST) and Serum Alanine Aminotransferase (ALT) activity were determined quantitatively by its enzymatic effect on the oxidation of NADH to NAD⁺.

Measurement of antibody titres of IgG, IgG1 and IgG2a against rHBsAg using indirect Enzyme-Linked Immunosorbent Assay (iELISA)

Analysis of specific total antibodies (IgG) were performed using an optimized indirect ELISA method described by Nejati et al. [16] on a 96-well ELISA plates (BRANDplates®, Germany) coated with 100 μL of rHBsAg. Similarly, the specific IgG1 and IgG2a subclasses were performed using goat anti- mouse IgG1 and IgG2a secondary antibodies (Southern biotechnology, USA).

Statistical analysis

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

Homology modelling of mucus protein and model validation

Structure prediction through homology modelling of the already deposited amino acid sequence of snail mucus protein on the NCBI protein database (Accession: QEG59314.1) was adopted to investigate the nature of the protein-protein interaction between the vaccine and the antigen [17,18]. The crystal structure of Mucus Proteins in garden snails was not available in the Protein Data Bank (PDB - www.rcsb.org), therefore, the 340 amino acids sequence of the mucus protein was subjected to 3D structure prediction using homology modelling strategy. A PDB blast analysis was carried out for the selection of model templates for the 340 amino acid sequence of snail mucus (Accession—QEG59313.1). The protein showing the highest sequence identity and query coverage was selected from the PDB-blast analysis and was used as a template for the model building of 3D structure of snail mucus protein on Swiss Model (https://swissmodel.expasy.org/interactive/) [19].

The predicted 3-D models were further refined with the GMQE (Global Model Quality Estimation) score assessment method in open sources Swiss Model package. The quality of the modelled structure with respect to stereo-chemical geometry and energy were analysed by SAVES v6.0 (https://saves.mbi.ucla.edu/) protein quality check package. The PDB structures of the snail mucus were also used for further protein interaction studies and in silico docking and dynamic studies.

Homology modelling of Hepatitis B surface antigen (HBsAg) and model validation

The complete crystal 3D structure of the envelope protein of Hepatitis B has not been deposited in the PDB (PDB - www.rcsb.org) although Li et al. [20] crystalized a partial sequence of the envelope protein known as Pres1. This study adopted homology modelling to obtain a model structure for the 385 amino acids sequence of the complete envelope protein of HBV, downloaded from the National Centre for Biotechnology Information (NCBI) protein database (www.ncbi.nlm.nih.gov/), having an accession number of AAK94656.1. The obtained model structure of HBsAg was also subjected to quality check on the SAVES v6.0 (https://saves.mbi.ucla.edu/) protein quality check package. The model structure was hence used for molecular docking studies and other dynamics in silico experiments.

Protein-protein alignment of snail mucus protein and HBsAg and generating structural morphs with PyMOL 2.4 version 2.4.1

Computational protein morphing produces an intermediate conformation resulting from the transitioning of protein conformations during protein-protein interaction. In this study, we used Morphing plugin in PyMOL 2.4 version 2.4.1 to generate a tentative morph structure, when a simulated interaction between the modelled HBsAg structure interacts with the modelled snail mucus protein. The generated morph was used for the docking experiment.

Studies of antigen (HBsAg)–antibody interaction using molecular docking

The modelled HBsAg 3D structure was docked to both the immunoglobulin structure against the antigen (PDB_ID– 5YAX) and the human immunoglobulin against HBV (PDB_ID– 4QGT). Similarly, the PDB file of the generated morph when snail mucus and antigen interact, was also docked with HBV antibody (PDB_ID– 5YAX) and human immunoglobulin (PDB_ID– 4QGT). The docking result was compared for both the modelled surface antigen (HBsAg) and the Morph structure.

Results

Snail mucin-adjuvanted rHBsAg vaccine had no adverse effects on experimental mice

To investigate if Snail Mucin as an adjuvant could pose some adverse effects on the animals, the body weight of the mice was measured throughout the study (day 0 to day 28). All five groups of mice had comparable growth patterns, inferring that there was no side effect caused by the administration of snail mucin or co-administration of snail mucin and vaccine on the animals (Fig 1).

Fig 1 — Results are expressed as mean ± SD (n = 5) with a confidence interval of 0.05.

Moreover, from the physical examination of livers, in all groups, the external surface of the liver was smooth, shiny, and reddish brown in colour suggesting healthy organs and slight variation in the liver weights was recorded among the groups (Fig 1). In summary, there was no mortality recorded in any experimental group throughout the experiment.

Snail mucin-adjuvanted rHBsAg vaccine had no significant toxicity in the liver

To evaluate if any form of liver toxicity did result from administering the Snail Mucin-Adjuvanted rHBsAg Vaccine. The activities of the liver-marker enzymes were measured. In the result shown in Table 1, the AST activity of the 2DVacSM experimental group was non-significantly (p>0.05) higher when compared to other experimental groups and the controls. Moreover, the value was within the normal range of AST for mice (54–298 U/L).

Table 1. Effect of snail mucin-adjuvanted rHBsAg vaccine on liver marker enzyme activities of experimental mice.

Group	AST (U/L)	ALT (U/L)
Group 1	147.99 ± 16.09^a	47.62 ± 5.18^a
Group 2	157.01 ± 13.49^a	41.71 ± 3.54^a
Group 3	160.92 ± 18.80^a	42.20 ± 4.92^a
Group 4	135.01 ± 12.05^a	45.58 ± 2.94^a
Group 5	140.51 ± 15.21^a	40.35 ± 2.27^a

Open in a new tab

Results are expressed as mean ± SD (n = 3). Mean values with different letters as superscripts along the columns were considered significant at p < 0.05.

Group 1: rHBsAg vaccine alone: 3 doses.

Group 2: rHBsAg vaccine alone: 2 doses.

Group 3: rHBsAg vaccine + Snail mucin: 2 doses.

Group 4: Snail mucin alone: 2 doses.

Group 5: Normal saline: 3 doses.

Similarly, the ALT activity of the 2DVacSM and 2DSM experimental group fell with the normal range of ALT enzyme activities for mice (17–77 U/L) and showed non-significantly (p>0.05) difference when compared to other groups and the control (Table 1).

Snail mucin-adjuvanted rHBsAg vaccine had no significant effect on the granulocyte count of experimental mice

Granulocytes consist of the Neutrophils, Eosinophils and Basophils. To observe the effect of Snail Mucin adjuvants on granulocytes, a differential white blood cell count was performed on day 0, 14, 21 and 28. The results from Fig 2 showed a steady significant (p<0.05) increase in the neutrophil count from day 0 to day 28 which was comparable among all experimental groups.

Fig 2 — Results are expressed as mean ± SD (n = 5) with a confidence interval of 0.05.

Furthermore, there were no significant (p>0.05) differences in the monocytes, eosinophils and basophils count among all experimental groups throughout the experiments (result not shown). Hence, there were no special effects caused by snail mucin on the neutrophils, monocytes, and eosinophils.

Effect of snail mucin-adjuvanted rHBsAg vaccine on cellular immune response (lymphocyte count) of experimental mice

To observe the effect of Snail Mucin-Adjuvanted rHBsAg Vaccine on lymphocytes, a differential white blood cell count was performed. There was a significant (p<0.05) decrease in Lymphocyte count for all experiment groups from day 0 to day 28 (Fig 3).

Fig 3 — Results are expressed as mean ± SD (n = 5) with a confidence interval of 0.05.

Effect of snail mucin-adjuvanted rHBsAg vaccine on the titre value of immunoglobulin G (IgG), immunoglobulin G subclass 1 (IgG1) and immunoglobulin G subclass 2a (IgG2a) of experimental mice

Indirect ELISA techniques were adopted to investigate the adjuvant effects of snail mucin on IgG, IgG1 and IgG2a titre. From the result as shown in Fig 4A, there was a significant (p<0.05) increase in the mean IgG titre of Group 3, co-administered with snail mucin and vaccine on day 28 (0.51±0.07) compared to before immunization (day 0–0.19±0.08), after prime immunization (day 14–0.19±0.02) and after boost immunization (day 21–0.27±0.03). Moreover, on day 28, the IgG titre of the group co-administered snail mucin and vaccine (Group 3) was significantly (p<0.05) higher than the negative controls (group 4 and 5 –administered with only mucin and normal saline) and the positive control (group 2 –administered with 2 doses of snail mucin).

Furthermore, there was a significant (p<0.05) increase in the mean IgG1 titre of the group co-administered snail mucin and vaccine on day 21 (0.46± 0.02) and day 28 (0.73±0.03) after boost immunization compared to day 0 (0.32±0.10) and day 14 (0.20±0.01), before and after prime immunization. Moreover, on day 28, the mean IgG1 titre of mice co-administered snail mucin and vaccine was found to be comparable to the groups administered 3 and 2 doses of vaccine alone and significantly (p<0.05) higher than the negative control (Fig 4B).

On the other hand, the titre results of the IgG2a from Fig 4C showed a contrasting trend when compared with IgG and subclass IgG1. There was a progressive and significant (p<0.05) decrease in mean IgG2a titre of the group co-administered snail mucin and vaccine after prime and boost immunizations [day 14 (0.13±0.03), day 21 (0.11±0.02) and day 28 (0.11±0.02)] compared to before immunization [day 0 (0.26±0.11)]. More so, the IgG2a titre of group 3 was comparable to the negative controls (group 4 and 5) and significantly lower (p<0.05) that the positive control.

Homology modelling of snail mucus protein and HBV envelope protein

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

The homology modelled snail mucus structure (Fig 5A) was interesting as it showed 8 Alpha helixes and 8 beta sheets. The local quality score for most of the residue was on average above 0.7 and the about 92.58% of the residues were favoured by the Ramachandran simulation. Finally, the modelled 3D structure of the protein gave a QMEAN score of above 0.5 with a standard deviation of the mean |Z-score| between 1 and 2.

Similarly, the structure of HBsAg (Fig 6A) was obtained through Homology Modelling of the amino acid sequence downloaded from the NCBI website (accession number—AAK94656.1) also on Swiss-Model (https://swissmodel.expasy.org/). A prior blast of the amino acid sequence against the Protein Data bank (PDB) gave a low query cover of 15%, but however, a 100% identity to the crystal structure (PDB ID - 5YAX_C) of a human neutralizing antibody against Hepatitis B virus, bound to a HBV preS1 peptide. On further analysis of the 5YAX crystal structure, the 100% identity was a result of the bounded Pres1 peptide.

Rheomorphic structural state from interaction of snail mucus protein and modelled HBsAg did not give a better docking score than the standalone HBsAg

A structural morph with about 30 rheomorphic states was generated from the alignment of modelled HBsAg and snail mucus protein using PyMOL v1.4.1. The generated morphs were docked to the immunoglobulin protein (PDB_ID– 5YAX) and the score was compared to the score from docking the standalone HBsAg to the same antibody (PDB_ID– 5YAX). The best predictions from the docking experiment, the morph, gave a lower negative score of -2646.06. Meanwhile, the best prediction from docking the standalone HBsAg gave a higher negative score of -4296.27. Although the score from the Morph docking was not better than the standalone antigen, it was discovered that both the Morph and the crystal Pres1 antigen interacted with the similar variable region for the Fab Fragment of the antibody (Fig 7), while the best prediction of the full HBsAg did not bind to the either variable region of the Fab fragments.

Fig 7 — a). The best prediction (-2646.06) for docking Morph to 5YAX. The brick red sphere represents the morph while the purple mesh are the surrounding interacting atoms on the variable region on the Fab segment of the heavy chain. The purple sphere represents the Pres1 peptide interacting with similar variable region of the light chain (blue mesh) of the Fab fragment. b). The best prediction when the standalone HBsAg envelope protein (the purple Helix-Turn-Helix Structure) is docked 5YAX. The green surface are residues of 5YAX interacting with the HBsAg which are not exactly the variable region of the heavy chain where antigen can bind. The red sphere is the Pres1 peptide interacting with residues on the light chain of 5YAX (blue surface).

Discussion

The toxicity profile and high cost of currently available vaccine adjuvants have necessitated the search for safer and more efficacious adjuvants for vaccines. Although the level of biotechnological advancement for generating vaccines have grew greatly with numerous recorded successes, there are no measurable development in the science of adjuvant, that may greatly enhance antibody responses to several bacterial and viral infection. The currently widely used hepatitis B vaccine antigen is a highly purified and derived from yeast-base recombinant technology. According to Schijns et al. [21], recombinant or purified vaccine antigens have poor immunogenicity due to removal of pathogen associated molecular patterns (PAMPs) which serve as intrinsic adjuvants; hence, they need other factors and components to boost their protective capacity against infections. These additional components are called adjuvants (from a Latin word “adjuvare” which means to help) are needed to enhance the immunogenicity of vaccine antigens improving the cumulative titre of antibody and effector T cells [21].

In the present study, vaccination of mice with recombinant hepatitis B surface antigen vaccine combined with snail mucin as well as with snail mucin alone did not result in growth retardation of the experimental animals. Experimental animals showed no difference in weight gain patterns compared to the controls. Although there were observed declines in body weights of all the animals after immunization and blood collection protocols because of handling stress, the animals recovered within a day or two. The observed normal growth pattern suggests that the treatments were safe.

Organ weight data are very useful for evaluation of toxicological change as a positive increase in liver weight can be accurately correlated with hepatocellular hypertrophy. To ensure proper experimental comparison and inference, Lazic, et al. [22] recommends that organ weight data are expressed as an organ-to-body weight ratio across the experimental groups. The mean liver weight of the group co-administered snail mucin and vaccine was comparable to the groups administered 3 and 2 doses of vaccine alone and the group administered normal saline. However, the liver tissues from the group administered snail mucin alone showed a significant increase in the mean weight as compared to the group administered normal saline. This increase in weight can be explained based on organ-to-body weight ratio. A higher body weight corresponds to a higher organ weight and vice versa. The mean body weight of the group administered snail mucin alone was higher than any of the other groups and so it is expected that the group should have the highest mean liver weight. Hence, there were no observed toxicological changes (hepatic hypertrophy) induced in the animals by immunization with snail mucin, rHBsAg vaccine or snail mucin-rHBsAg vaccine. This is further supported by the external morphological features of the organs which suggested that the organs were healthy. In all the groups, the external surface of the liver was smooth, shiny, and reddish brown in colour. These are the characteristics of normal, healthy livers. Similarly, Yuan et al. [23] adopted the use of the body weight of chickens to evaluate if any toxicity arose from the use of sunflower seed oil and ginseng stem-leaf saponins when used as an adjuvant for Newcastle disease vaccine.

To further ascertain that there was no hepatotoxicity caused by the vaccine formulation, serum transaminase levels were investigated. Following onset of liver enlargement, the increase in hepatic-derived enzymes (transaminases, alkaline phosphatase, and γ-glutamyltransferase) are frequently observed [24]. Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST) activities are vital pathology parameters helpful in the investigation of adverse effects of drugs/chemicals on the liver. Increases in serum ALT activity are thought to be due to damage to, and leakage from hepatocyte cell membranes, resulting in a release of enzymes from the cytosol into the blood [25]. Increases in ALT activity of two to three folds are indicative of hepatocellular damage. The AST and ALT levels in the group co-administered snail mucin and vaccine were found to be comparable to the negative control group. They were within the normal acceptable range for mice (17–77 U/L for ALT and 54–298 U/L for AST). Hence, there was no indication of hepatotoxicity caused by the vaccine formulation.

Recent empirical data suggest that many adjuvants enhance T and B cell responses by interacting with several components of the innate immune system, rather than by direct effects on the lymphocytes themselves [26]. This was the rationale behind the differential white blood cell count performed in the present study. Four of the five white blood cell types identified during the differential WBC count (neutrophils, monocytes, eosinophils, and basophils) are components of the innate immune system.

The increase in the monocyte count of the group co-administered snail mucin and vaccine compared to the groups administered 3 and 2 doses of vaccine alone on day 14 is evidence of immune stimulatory capacity of the formulation. Monocytes, which differentiate into macrophages and dendritic cells upon maturity, are highly important in activating adaptive immune responses. They are highly important in phagocytosis, antigen presentation, cytokine production, and antimicrobial and cytotoxic activities [27]. Thus, stimulation of monocytes is an indication that snail-mucin adjuvanted rHBsAg vaccine is more immunogenic than the vaccine alone.

No significant basophil release was detected in any of the groups. This is in line with O’Connell et al. [28], who reported that basophils are rarely present in murine peripheral blood. The absence of basophils in the group co-administered snail mucin and vaccine proves that the vaccine formulation did not produce any allergic reaction.

There was an upregulation of eosinophils in the group co-administered snail mucin and vaccine on day 21 compared to the groups administered 3 and 2 doses of vaccine alone. Although eosinophils are more important in resistance to parasites, they are phagocytic cells, and their upregulation suggests innate immune system activation. According to Iwasaki and Medzhitov [27], eosinophils are important to the innate response and help to link the innate immune response to the adaptive immune response.

Neutrophils are largest in number of all other leukocytes and functions as the critical effector cells of the innate immune system [29]. Neutrophils expresses a broad pattern of recognition receptors such as toll-like receptors, Fc receptors and complement components. Majorly, neutrophil annihilate microorganisms through phagocytosis, release of cytotoxic granules and use of neutrophil extracellular traps [30] and hence, they are important innate response that coordinate to improve adaptive immune responses [31]. Several studies have proven the ability of mouse neutrophils to serve as antigen presenting cells (APCs) or influence the capacity of professional APCs to present antigens [32]. Vono et al. [32] showed that neutrophils can present cognate antigens to antigen-specific memory CD4+ T cells. And hence, they are invaluable in the immunity against disease.

Co-administration of snail mucin with vaccine caused significant increases in neutrophil counts compared to administration of vaccine alone (Fig 2). There was a corresponding decrease in lymphocyte count. This is in consonance with Provencher et al. [33] who stated that in mice, lymphocyte counts decrease as neutrophil counts increase. The observed increase in neutrophil count indicates that neutrophils probably functioned as APCs, leading to increased activation of antigen-specific B lymphocytes as observed from the data on humoral immune responses.

The mean IgG titre of the group co-administered snail mucin and vaccine showed a non-significant (p>0.05) increase on day 14 after prime immunization. This could be explained by the fact that adaptive immune responses are activated after boost immunization. After the boost immunization, there was also a non-significant (p>0.05) increase in the mean IgG titre observed on day 21. This non-significant increase could be attributed to the possible entrapment of the vaccine antigen by snail mucin microparticles. Polymeric biodegradable microparticles used as vaccine adjuvants have been shown to entrap antigen and release it gradually over a period. The advantage of this mechanism is that it mimics the prime and boosts doses of vaccination, and therefore elicits robust immune responses in a single administration [34].

The delivery of antigens loaded on polymeric particle systems has several advantages: it prolongs antigen presence, enhances DC-mediated antigen uptake, directs stimulation of DCs, and promotes longer and better immune responses [35]. The proposed antigen entrapment mechanism was further supported by a marked significant (p>0.05) increase in the mean IgG titre of the group co-administered snail mucin and vaccine compared to the group administered 2 doses of the vaccine alone on day 28 (Fig 4A). However, there was a decrease (non-significant) in the mean IgG titre of the group administered 2 doses of the vaccine alone (group 2) on day 28, suggesting antigen clearance and termination of immune stimulation. Snail mucin serves as a biodegradable polymer which generally have great application in medicines. They are used to deliver drug and vaccine into the body are they are enzymatically hydrolysed in the body releasing their content [36].

The significant increases in the mean IgG1 titres of the group co-administered snail mucin and vaccine on days 21 and 28 compared to day 14 (Fig 4) can be explained by entrapment and sustained release of antigen by adjuvant (snail mucin), resulting in continuous and persistent stimulation of the immune system over a period of time such that by day 28, the mean IgG1 titre of the group co-administered snail mucin and vaccine (2 doses) was comparable to the group administered 3 doses of vaccine alone. The increased IgG1 titre indicates a strong Th2 type (humoral) response.

Cytotoxic T-lymphocytes (CTLs) stimulated by Th1 response is not essential for protective immunity against HBV [37]. IgG2a stimulates Th1 responses while IgG1 stimulates Th2 responses. This is a possible explanation for the trend of results obtained for mean IgG2a titre (Fig 4). The group co-administered snail mucin and vaccine had comparable IgG2a titres with groups administered 2 doses of vaccine alone, snail mucin alone and normal saline. Another possible explanation for this observation is that the strain of mice used for the study may not be expressing IgG2a. Shinde et al. [38] reported that in some strains of mice there is no expression of IgG2a; instead these mice express the novel IgG2c. Thus, it can be inferred that IgG2a is not a useful immunoglobulin subclass to monitor immune responses to recombinant hepatitis B surface antigen vaccine in non-IgG2a expressing mice.

The homology modelled snail mucus structure (Fig 5A) and the complete envelope protein of the HBV (HBsAg) was modelled on Swiss-Model program using the downloaded amino acid sequence from the NCBI protein database (Accession number—AAK94656.1). Although, the complete envelope structure has not been deposited in the protein data bank, one study conducted by Li et al. [20] successfully elucidated the X-ray crystal structure of a peptide which is part of the envelope protein of the HBV and responsible for virulence (Pres1 peptide). The structure of the Pres1 peptide was deposited on the PDB database when bonded to a human neutralizing antibody against HBV infection [20]. Pres 1 peptide is part of the envelope protein that is responsible for the virulence of HBV. Pres1 binds to the Sodium Taurocholate Cotransporting Polypeptide (NTCP) of the hepatocytes, hence resulting in Hepatitis [22].

Results from the docking experiment showed the standalone protein had a better docking score than morph when the best prediction out of 100 was compared. However, when the structure of the best prediction was observed closely with PyMOL v2.4.1 and VMD (Fig 7A and 7B), the morph interacted with mainly with the variable region of the Fab fragment of 5YAX while the standalone protein interacted with other residues other than the variable region. The antibody (immunoglobulin) has a consistent structure constituting two the heavy chain and two light chains, which are further grouped into the two Fab (fragment of antigen binding) and the one Fc (Fragment crystallizable) [39]. The specificity of antibody to a single antigen is brought about by the variable segment of the Fab region. So, if the interaction between the standalone antigen and the antibody occurs around residues away from the variable Fab segment, it implies that there might be an interaction which is not a favourable antigen-antibody complex. Interaction of the standalone HBsAg with snail mucus generated several rheomorphic structures that interacted with the variable region of the Fab fragment, although with a lower docking score.

Conclusion

Based on this study, snail mucin could serve as an effective and safe adjuvant for recombinant hepatitis B surface antigen vaccine. It is well-tolerated and elicits appropriate immune responses. It probably exerts its effects by sustained and gradual release of vaccine antigen thereby producing continuous and persistent stimulation of immune responses. The proposed mechanism mimics the prime and booster doses of recall immunizations and directly translates into reduction of the number of doses/injections needed to attain protective antibody levels. One promising benefit of such is single-administration immunization programs and cost effectiveness.

Supporting information

S1 Table. Effect of hepatitis-B vaccine modulated with Egg Shell membrane on eosinophil count.

(PDF)

Click here for additional data file.^{(113.7KB, pdf)}

S2 Table. Effect of snail mucin-adjuvanted rHBsAg vaccine on monocyte count of experimental mice.

(PDF)

Click here for additional data file.^{(113.8KB, pdf)}

S3 Table. Effect of snail mucin-adjuvanted rHBsAg vaccine on basophil count of experimental mice.

(PDF)

Click here for additional data file.^{(112.8KB, pdf)}

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

The author(s) received no specific funding for this work.

References

1.Sivakumar SM, Safhi MM, Alam MF, Anwer T, Khan G, Jabeen A. Insight on hepatitis B vaccine adjuvanticity “future perspectives.” SM J Hepat Res Treat. 2016;2: 1007. [Google Scholar]
2.Gebrecherkos T, Girmay G, Lemma M, Negash M. Knowledge, Attitude, and Practice towards Hepatitis B Virus among Pregnant Women Attending Antenatal Care at the University of Gondar Comprehensive Specialized Hospital, Northwest Ethiopia. Int J Hepatol. 2020;2020. doi: 10.1155/2020/5617603 [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Zheng Y, Wu J, Ding C, Xu K, Yang S, Li L. Disease burden of chronic hepatitis B and complications in China from 2006 to 2050: An individual-based modeling study. Virol J. 2020;17: 1–10. doi: 10.1186/s12985-019-1274-x [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Di Pasquale A, Preiss S, Da Silva FT, Garçon N. Vaccine adjuvants: From 1920 to 2015 and beyond. Vaccines. 2015;3: 320–343. doi: 10.3390/vaccines3020320 [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Djurisic S, Jakobsen JC, Petersen SB, Kenfelt M, Klingenberg SL, Gluud C. Aluminium adjuvants used in vaccines. Cochrane Database Syst Rev. 2018;2018. doi: 10.1002/14651858.CD013086 [DOI] [Google Scholar]
6.Meireles LC, Marinho RT, Van Damme P. Three decades of hepatitis B control with vaccination. World J Hepatol. 2015;7: 2127–2132. doi: 10.4254/wjh.v7.i18.2127 [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Zimmermann P, Curtis N. Factors That Influence the Immune Response to Vaccination. Am Soc Microbiol. 2019;32: 1–50. doi: 10.1128/CMR.00084-18 [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Arhewoh MI, Eraga SO, Builders PF, Ibobiri MA. Effect of mucin extraction method on some properties of metronidazole mucoadhesive loaded patches. J Pharm Bioresour. 2015;12: 1–7. [Google Scholar]
9.Harti AS, Sulisetyawati SD, Murharyati A, Oktariani M. The effectiveness of snail slime and chitosan in wound healing. Int J Pharma Med Biol Sci. 2016;91: 2–6. [Google Scholar]
10.Momoh MA, Chime SA, Ogbodo DU, Akudike PK, Udochukwu SU, Ossai EC, et al. Biochemical, rheological and hydrophile-lipophile balance (HLB) evaluation of archachatina marginata (snail) mucin extract for possible nutraceutical and nano biopharmaceutical applications. Trop J Pharm Res. 2019;18: 927–934. doi: 10.4314/tjpr.v18i5.3 [DOI] [Google Scholar]
11.Kalu AA, Ogbonnaya E, Rabiu KM, Uche EM, Zabaratu Y. Effect of Archachatina marginata mucin on the aggressive factors of gastric ulcer challenged wistar rat stomach tissue. GSC Biological and Pharmaceutical Sciences. 2019;9(1): 077–082. doi: 10.30574/gscbps.2019.9.1.0163 [DOI] [Google Scholar]
12.Adikwu MU, Nnamani PO. Some physiological and toxicological properties of snail mucin. Bioresearch. 2005;5: 1–6. [Google Scholar]
13.Carr JH, Rodak BF. Clinical Hematology Atlas 3rd Edition. Saunders: Elsevier. 2009. [Google Scholar]
14.Thomas L. Alanine aminotransferase (ALT), Aspartate aminotransferase (AST). in Clinical Laboratory Diagnostics. 1st ed. Thomas L, editor. Frankfurt: TH-Books Verlagsgesellschaft; 1998. [Google Scholar]
15.Moss DW, Henderson AR. Clinical enzymology. 3rd ed. Burtis CA, Ashwood ER, editors. Philadelphia: Tietz Textbook of Clinical Chemistry; 1999. [Google Scholar]
16.Nejati S, Mirzaee S. N, Nouri HR, Farhoudi R, Namvar NA, Yazdi MH, et al. Immune responses of mice immunized with HBsAg formulated in naloxone/alum mixture: comparison to Fendrix vaccine. Hepat Mon. 2017;17: 44536. [Google Scholar]
17.O’Leary NA, Wright MW, Brister JR, Ciufo S, Haddad D, McVeigh R. et al. Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional annotation. Nucleic Acids Res. 2016;44: D733–745. doi: 10.1093/nar/gkv1189 [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Launay G, Simonson T. Homology modelling of protein-protein complexes: A simple method and its possibilities and limitations. BMC Bioinformatics. 2008;9: 1–16. doi: 10.1186/1471-2105-9-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Waterhouse A., Bertoni M, Bienert S, Studer G, Tauriello G, Gumienny R, et al. SWISS-MODEL: homology modelling of protein structures and complexes homology modelling of protein structures and complexes. Nucleic Acids Res. 2018;46: W296–W300. doi: 10.1093/nar/gky427 [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Li D, He W, Liu X, Zheng S, Qi Y, Li H, et al. A potent human neutralizing antibody Fc-dependently reduces established HBV infections. Elife. 2017;6: e26738. doi: 10.7554/eLife.26738 [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Schijns V, Fernández-Tejada A, Barjaktarović Ž, Bouzalas I, Brimnes J, Chernysh S, et al. Modulation of immune responses using adjuvants to facilitate therapeutic vaccination. Immunol Rev. 2020;296: 169–190. doi: 10.1111/imr.12889 [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Lazic SE, Semenova E, Williams DP. Determining organ weight toxicity with Bayesian causal models: Improving on the analysis of relative organ weights. Sci Rep. 2020;10: 1–12. doi: 10.1038/s41598-019-56847-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Yuan L, Wang Y, Ma X, Cui X, Lu M, Guan R, et al. Sunflower seed oil combined with ginseng stem-leaf saponins as an adjuvant to enhance the immune response elicited by Newcastle disease vaccine in chickens. Vaccine. 2020;38: 5343–5354. doi: 10.1016/j.vaccine.2020.05.063 [DOI] [PubMed] [Google Scholar]
24.McGill MR. The past and present of serum aminotransferases and the future of liver injury biomarkers. EXCLI J. 2016;15: 817–828. doi: 10.17179/excli2016-800 [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Shibabaw T, Dessie G, Molla MD, Zerihun MF, Ayelign B. Assessment of liver marker enzymes and its association with type 2 diabetes mellitus in Northwest Ethiopia. BMC Res Notes. 2019;12: 1–5. doi: 10.1186/s13104-018-4038-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Nanishi E, Dowling DJ, Levy O. Toward precision adjuvants: Optimizing science and safety. Curr Opin Pediatr. 2020;32: 125–138. doi: 10.1097/MOP.0000000000000868 [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Iwasaki A, Medzhitov R. Control of adaptive immunity by the innate immune system. Nat Immunol. 2015;16: 343–353. doi: 10.1038/ni.3123 [DOI] [PMC free article] [PubMed] [Google Scholar]
28.O’Connell KE, Mikkola AM, Stepanek AM, Vernet A, Hall CD, Sun CC, et al. Practical murine hematopathology: A comparative review and implications for research. Comp Med. 2015;65: 96–113. [PMC free article] [PubMed] [Google Scholar]
29.Selders GS, Fetz AE, Radic MZ, Bowlin GL. An overview of the role of neutrophils in innate immunity, inflammation and host-biomaterial integration. Regen Biomater. 2017;4: 55–68. doi: 10.1093/rb/rbw041 [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Teng TS, Ji AL, Ji XY, Li YZ. Neutrophils and immunity: From bactericidal action to being conquered. J Immunol Res. 2017;2017. doi: 10.1155/2017/9671604 [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Irvine DJ, Aung A, Silva M. Controlling timing and location in vaccines Darrell. Adv Drug Deliv Rev. 2020;158: 91–115. doi: 10.1016/j.addr.2020.06.019 [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Vono M, Lin A, Norrby-Teglund A, Koup RA, Liang F, Loré K. Neutrophils acquire the capacity for antigen presentation to memory CD4+ T cells in vitro and ex vivo. Blood. 2017;129: 1991–2001. doi: 10.1182/blood-2016-10-744441 [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Provencher BA, Everds NE, Zimmerman KL, Moore DM, Smith SA, Barnhart KF. Hematology of laboratory animals. 1st ed. Weiss D, Wardrop J, Chalm OW, editors. Hoboken (NJ): Wiley-Blackwell: Schalm’s Veterinary Hematology; 2010. doi: 10.1258/la.2010.009049 [DOI] [Google Scholar]
34.Lin C-Y, Lin S-J, Yang Y-C, Wang D-Y, Cheng H-F, Yeh M-K, et al. Human Vaccines & Immunotherapeutics Biodegradable polymeric microsphere-based vaccines and their applications in infectious diseases Biodegradable polymeric microsphere-based vaccines and their applications in infectious diseases. 2015;11: 650–656. Available: http://www.tandfonline.com/action/journalInformation?journalCode=khvi20%5Cn 10.1080/21645515.2015.1009345. [DOI] [PMC free article] [PubMed] [Google Scholar]
35.Joshi VB, Geary SM, Salem AK. Biodegradable particles as vaccine antigen delivery systems for stimulating cellular immune responses. Hum Vaccines Immunother. 2013;912: 2584–2590. doi: 10.4161/hv.26136 [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Gutjahr A, Phelip C, Coolen AL, Monge C, Boisgard AS, Paul S, et al. Biodegradable polymeric nano particles-based vaccine adjuvants for lymph nodes targeting. Vaccines. 2016;4: 34. [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Kondo Y, Ninomiya M, Kakazu E, Kimura O, Shimosegawa T. Hepatitis B Surface Antigen Could Contribute to the Immunopathogenesis of Hepatitis B Virus Infection. ISRN Gastroenterol. 2013;2013: 1–8. doi: 10.1155/2013/935295 [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Shinde P, Howie HL, Stegmann TC, Hay AM, Waterman HR, Szittner Z, et al. IgG Subclass Determines Suppression Versus Enhancement of Humoral Alloimmunity to Kell RBC Antigens in Mice. Front Immunol. 2020;11: 1516. doi: 10.3389/fimmu.2020.01516 [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Chiu ML, Goulet DR, Teplyakov A, Gilliland GL. Antibody Structure and Function: The Basis for Engineering Therapeutics. Antibodies. 2019;8: 55. doi: 10.3390/antib8040055 [DOI] [PMC free article] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0246915.r001

Decision Letter 0

Isabelle Chemin

1 Apr 2021

PONE-D-21-02861

Experimental and Molecular Predictions of the Adjuvanticity of Snail Mucin on Hepatitis B Vaccine in Albino Mice

PLOS ONE

Dear Dr. Ezeorba,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

In particular the Figure 4 results that is pivotal in the paper's conclusions and statements. Methodological approaches are not sound and well described.

Please submit your revised manuscript by 2 months. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Isabelle Chemin, PhD

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

In your Methods section, please state the volume of the blood samples collected for use in your study.

To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information on the animal research and ensure you have included details on (1) methods of sacrifice, (2) methods of anesthesia and/or analgesia, and (3) efforts to alleviate suffering.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The current manuscript authored by Joshua et al., focuses the study of adjuvanticity of snail mucin with recombinant Hepatitis B Vaccine 28 (rHBsAg). The authors state that this study has shown that snail mucin possesses some adjuvant properties and enhances immune response towards rHBsAg vaccine. This is an interesting report. However, the data is not convincing, especially the data in Figure 4. The conclusions drawn are not supported by the experimental data. This manuscript in its present form does not meet the Plos One publication standard. I would ask the authors to address several key issues for further consideration.

Major concerns:

1. In figure 4, which is the most important figure in this manuscript, the authors tried to make a statement that 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. This statement is not true and is not based on the experiment result. Group 3 could be significantly higher than Group 5, the negative controls. However, the most important comparison is between Group 2 ( rHBsAg vaccine alone: 2 doses) and Group 3 ( rHBsAg vaccine + Snail mucin: 2 doses). There was no time point that showed that Group 3 is significantly better than Group 2, in terms of IgG, IgG1 or IgG2 titres.

2. The authors should consider demonstrating IgG, IgG1 or IgG2 of anti-HBsAg antibodies in Figure 4 in separate panels. For example, one panel shows IgG titer only in all time point. It is crucial to add statistics results (error bars) to each of the figures.

3. Snail mucin toxicity needs to be determined by a dosage test. After the animals are treated with increasing amount of Snail mucin, the body weight of the mice will be measured daily to determine the minimum amount of Snail mucin that causes adverse effects on the animals.

4. A survival test is strongly recommended to support the conclusion. Lethal dose (LD50 is recommended) of Hepatitis B will be given to all groups of the mice after they received their treatment on Day 28. If the mucin is helpful as adjuvants, we will find more mice in Group 3 survive the lethal virus challenge than the mice in Group 2.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2021 Jul 23;16(7):e0246915. doi: 10.1371/journal.pone.0246915.r002

Author response to Decision Letter 0

27 May 2021

Dr. Isabelle Chemin,

Academic Editor,

PLOS ONE Journal.

Dear Dr. Isabelle,

RESPONSE TO ACADEMIC EDITOR AND REVIEWER

I sincerely appreciate the privilege of reviewing our research article entitled “Experimental and Molecular Predictions of the Adjuvanticity of Snail Mucin on Hepatitis B Vaccine in Albino Mice”. Thank you very much for your comments and feedback, and also that of the reviewer. I have thoroughly effected all possible corrections and made relevant additions based on your feedback. I sincerely hope you find them meritorious for your reputable Journal (PLOS ONE).

Based on the Academic Editor comments which focused on the journal requirements, here are my responses and corrections that have been effected on the manuscripts

1. I ensured that the manuscript now meets the PLOS ONE’s style requirements, especially with regards to proper figure and table labelling, using the right font size for level 1 and 2 headings and using upper cases only for the first letter of all headings.

2. In my first submission, I erroneously omitted the volume of blood samples used for the study in the method section. However, in this revised manuscript, the volume of blood collected, which was 0.5 mL, has been included in the method section.

3. Finally, the methods on handling the animals have been explicitly included in the revised manuscript. The method of cervical dislocation was adopted for animal euthanasia by a well-trained researcher, who ensured that the suffering of the animals were reduced to the barest minimum. We did not administer anaesthesia or analgesia to the animals despite their pain-relieving activities, to avoid interference with other intended histopathology analysis. More so, snail mucin has been reported to possess some anaegestic properties, hence possibly reducing the pain in the animals (Adikwu and Nnamani, 2005).

Based on the peer-reviewer’s major concerns, here are my responses and corrections that have been effected on the manuscripts.

Major concerns 1 and 2:

I sincerely appreciate all the comments of the reviewer.

Fig 4 from my initial submission was clumsy. It was a composite graph showing the IgG, IgG1 and IgG2 titre values together, for different time point. Hence, the figure did not explicitly despite some of the claims from my results.

To address this issue based on the reviewer suggestion, I have separated the composite graphs into three separate graphs. The new graphs (Fig 4a, 4b and 4c) now show the mean titre value for IgG, IgG1 and IgG2 with their various statistics (error bar) respectively.

The reviewer pointed out that my claim of observing a significantly (P<0.05) higher mean titre of anti-HBsAg bodies in group 3 (administered with mucin and rHBsAg) when compared to negative control (group 5) was correct. However, my result didn’t support the assertion of a high antibody titre in group 3 when compared to the positive control (group 2-adminstered with rHBsAg only) at any time point.

The new Fig 4a showed that on day 28, a significantly higher IgG titre value of the experimental group 3 (administered with 2 doses of the vaccine and mucin), when compared with both the positive control group 2 (administered only with 2 doses of rHBsAg vaccine only) and the negative control group 4 and 5 (administered with only mucin and normal saline respectively).

Although, the result on day 14 and 21 did not show significant (P>0.05) differences in the IgG titre across the treatment group 3 and control group 2 & 5, as compared to what was observed on day 28. This result patterned our thinking to the possible mechanisms of adjuvanticity of snail mucin on rHBsAg, as was explained in the discussion section.

We explained that the mucin, possibly acts as a Polymeric Biodegradable Microparticles, entrapping the antigen and releasing it gradually over a period of time. The proposed antigen entrapment mechanism was supported by a marked significant (p>0.05) increase in the mean IgG titre of group 3 (co-administered snail mucin and vaccine) compared to group 2 (administered 2 doses of the vaccine alone) on day 28 (Fig 4a).

Similarly, the new Fig 4b, showed a very interesting result. Although the IgG1 titre of the treatment group 3 was significantly lower than the positive control group 2 on day 21 and non-significantly lower on day 28, there was a relatively significant boost in the IgG1 titre for the treatment group from day 21 to day 28, while the positive control group 2 showed a decrease in IgG1 titre from day 21 to day 28. This further supports our assumptions of possible antigen entrapping mechanism of Snail Mucin.

Lastly, the results of the new Fig 4c showed a contrasting trend for the mean titre value of IgG2. Studies have shown that Cytotoxic T-lymphocytes (CTLs) stimulated by Th1 response are not essential for protective immunity against HBV (Konda et al., 2013). It is known that IgG2a stimulates Th1 responses while IgG1 stimulates Th2 responses. Hence, the possible explanation for a low IgG2 titre across the group.

Major concerns 3:

The reviewer pointed out the need for toxicity studies which is very relevant for determining the effective dosage that can be accommodated in the animals.

In this present study, it is true we did not perform any toxicity studies on the mice, despite its significance. However, our choice for the dose formulation for snail mucin was not done blindly but was guided by a study conducted by Adikwu and Nnamani (2005). It was reported in that study that snail mucin was well tolerated, as the animals (mice 20-25 g) did not show any symptoms of toxicity and none of the animals died (Adikwu and Nnamani, 2005). Since, a similar experiment on the animals has been published in the past, we decided not to repeat the same experiments, with the aim of avoiding the unjustifiable use of animals for research - based on our institutional animal laws.

Moreover, our results from our study showed that snail mucin was non-toxic to the animals as, no animal died during the study, no significant changes in the weight of the animal and no obvious allergic reaction was observed at the point of administration. Finally, the liver weight and histology were similar for all the experimental groups.

Major concerns 4:

The reviewer recommended that a survival test study be conducted to fully support the conclusions that snail mucin is helpful as an adjuvant for rHBsAg vaccine.

It is true that we did not conduct a survival test, during the experimental phase of the studies. This study is relevant as it may give more confirmatory evidence to our assertion that snail mucin has some adjuvant properties in rHBsAg. However, our conclusion was based on general scientific knowledge about the correlation of elevated antibody titres with vaccine efficacy. Moreover, the survival test is imperative to determine whether these antibodies generated could really neutralize the virus.

The results we have presented have shown that snail mucin has some possible adjuvant properties, through the possible Polymeric Biodegradable entrapment mechanism – entrapping the antigen, releasing it in bits to sustained last longing immunity, and reducing the need for multiple vaccinations.

Despite the relevance of this suggested study (survival test), it may be very difficult to perform this experiment now as we have euthanized all the animals used for the studies. More so, culturing HBV for infecting the animal is very difficult due to restrictions within the institution when working with infectious viral particles. Regardless, this will be done in a future study, when we secure collaborations with a virology institute/laboratory.

Thank you very much for taking the time to go through my responses. Hope you find them worthy.

Yours sincerely,

Timothy P. C. Ezeorba

Timothy.ezeorba@unn.edu.ng

Attachment

Submitted filename: Response to Reviewer.pdf

Click here for additional data file.^{(239.9KB, pdf)}

PLoS One. doi: 10.1371/journal.pone.0246915.r003

Decision Letter 1

Isabelle Chemin

17 Jun 2021

PONE-D-21-02861R1

Experimental and Molecular Predictions of the Adjuvanticity of Snail Mucin on Hepatitis B Vaccine in Albino Mice

PLOS ONE

Dear Dr. Ezeorba

Please submit your revised manuscript by 4 weeks. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Isabelle Chemin, PhD

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

**********

6. Review Comments to the Author

Reviewer #1: I want to thank the authors for re-submitting the article "Experimental and Molecular Predictions of the Adjuvanticity of Snail Mucin on Hepatitis B Vaccine in Albino Mice". The modifications made to this paper have greatly improved its quality.

I have 2 minor remarks for this manuscript:

- Can the authors explain why Group 2 is similar to Group 4 (Snail Mucin only, no vaccine) on Day 28 in Fig.4a?

-A figure legend usually has a title, and Materials and method (A description of the techniques used). P-values and the sample size, if applicable, should also be included. Please complete all figure legends in the manuscript.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

PLoS One. 2021 Jul 23;16(7):e0246915. doi: 10.1371/journal.pone.0246915.r004

Author response to Decision Letter 1

7 Jul 2021

University of Nigeria, Nsukka

Faculty of Biological Sciences

Department of Biochemistry

Timothy P. C. Ezeorba 24th May 2021

(Lecturer II)

Dr. Isabelle Chemin,

Academic Editor,

PLOS ONE Journal.

Dear Dr. Isabelle,

RESPONSE TO ACADEMIC EDITOR AND REVIEWER

Once again, I sincerely appreciate the privilege of reviewing our research article entitled “Experimental and Molecular Predictions of the Adjuvanticity of Snail Mucin on Hepatitis B Vaccine in Albino Mice”. Thank you very much for your comments and feedback, and also that of the reviewer. I sincerely hope you find this rebuttal meritorious for your reputable Journal (PLOS ONE).

Review based on the journal requirements

I have reviewed the reference list and ensured that they are now appropriate. I replaced Ref number [11], Adikwu and Ikejiuba, (2005) with a new reference (Kalu et al., 2019).

Reason - I discovered that the journal website where Adikwu and Ikejiuba, (2005) published have been pulled out and is no longer accessible. The new reference (Kalu et al., 2019) is more recent and addresses similar methods and ideas as the previous reference used.

To the best of my knowledge, other references are appropriate for publication.

Based on the peer-reviewer’s major concerns, here are my responses and corrections that have been effected on the manuscripts.

I want to sincerely appreciate the reviewer for the detailed and thorough review of this work. I have ensured to respond appropriate to the reviewer’s comments to the best of my ability. Presented below are the reviewer’s comments and my responses.

- Can the authors explain why Group 2 is similar to Group 4 (Snail Mucin only, no vaccine) on Day 28 in Fig.4a?

Thank you very much for pointing that out from our result as depicted in Figure 4a.

The results on Figure 4a show the IgG titre value measured using iELISA for all the experimental groups from day 0 to 28. The results on day 28 showed a similar IgG titre value for animals in group 2 (administered 2 doses of the vaccine alone) and group 4 (a negative control administered 2 doses of mucin alone), as pointed out by the reviewer. Intuitively, one may think that there should be a significant difference among the two groups, however, a non-significant (P>0.05) increase in group 2 as compared to group 4 was observed. The possible reasons for an increased IgG in the group 4 animals may be due to immunogenic responses in some of the animals within the group which may have been challenged with antigens within their environment (inhaled or ingested). This may be evident as pointed out by statistical analysis which showed a considerably higher error bar in group 4 on day 28, when compared to other experimental groups.

Furthermore, there are possibilities that snail mucin, since foreign to the animal, may have resulted in a level of antibody production, accounting for the IgG titre in group 4. Although there are very limited studies on the immunogenicity of snail mucins, few studies available highlighted contrasting opinions. A study conducted by Karne et al. (2017) showed mucin protein from Macrochlymus indica (a species of Terrestrial Land Snail) to possess some immunogenic properties after an IELISA experiment. While, another study conducted by Harti et al. (2018) pointed out that snail mucus from Achatina fulica on its own could not stimulate lymphocyte proliferation. Hence, there is need for future studies to investigate thoroughly the immunogenic potential of Snail mucin from different varieties of snails.

- A figure legend usually has a title, and Materials and method (A description of the techniques used). P-values and the sample size, if applicable, should also be included. Please complete all figure legends in the manuscript.

Thank you very much for the comments.

It is very true that a description for each of the figures are necessary. I have done that in my previous submission. The figure legends were place in the body of the article, at a position where the Figures should be placed. This was in line with the journal format for submission.

However, I have improved the legends, by adding some of its vital components as you pointed out from your comment. Thank you.

Thank you very much for taking the time to go through my responses. Hope you find them worthy.

Your sincerely,

Timothy, P. C. Ezeorba

Timothy.ezeorba@unn.edu.ng

References

Karne S. P., Gupta A., Sumesh S., kamble, S. and Bharat S. (2017). Exploration of mucin protein from Macrochlymus indica and determined its immunogenicity studies. Journal of Biomedical and Pharmaceutical Research, 6(1): 33-38. Retrieved from https://jbpr.in/index.php/jbpr/article/view/83

Harti, A. S., A. Murharyati, D. S. S, and M. Oktariani (2018). The effectiveness of snail mucus (Achatina fulica) and chitosan toward limfosit proliferation in vitro. Asian Journal of Pharmaceutical and Clinical Research 11(15): 85-88, doi:10.22159/ajpcr.2018.v11s3.30041

Attachment

Submitted filename: Rebuttal letter.pdf

Click here for additional data file.^{(307.2KB, pdf)}

PLoS One. doi: 10.1371/journal.pone.0246915.r005

Decision Letter 2

Isabelle Chemin

12 Jul 2021

Experimental and Molecular Predictions of the Adjuvanticity of Snail Mucin on Hepatitis B Vaccine in Albino Mice

PONE-D-21-02861R2

Dear Dr. Ezeorba,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Isabelle Chemin, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0246915.r006

Acceptance letter

Isabelle Chemin

15 Jul 2021

PONE-D-21-02861R2

Experimental and Molecular Predictions of the Adjuvanticity of Snail Mucin on Hepatitis B Vaccine in Albino Mice

Dear Dr. Ezeorba:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Mrs Isabelle Chemin

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. Effect of hepatitis-B vaccine modulated with Egg Shell membrane on eosinophil count.

(PDF)

Click here for additional data file.^{(113.7KB, pdf)}

S2 Table. Effect of snail mucin-adjuvanted rHBsAg vaccine on monocyte count of experimental mice.

(PDF)

Click here for additional data file.^{(113.8KB, pdf)}

S3 Table. Effect of snail mucin-adjuvanted rHBsAg vaccine on basophil count of experimental mice.

(PDF)

Click here for additional data file.^{(112.8KB, pdf)}

Attachment

Submitted filename: Response to Reviewer.pdf

Click here for additional data file.^{(239.9KB, pdf)}

Attachment

Submitted filename: Rebuttal letter.pdf

Click here for additional data file.^{(307.2KB, pdf)}

Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

[pone.0246915.ref001] 1.Sivakumar SM, Safhi MM, Alam MF, Anwer T, Khan G, Jabeen A. Insight on hepatitis B vaccine adjuvanticity “future perspectives.” SM J Hepat Res Treat. 2016;2: 1007. [Google Scholar]

[pone.0246915.ref002] 2.Gebrecherkos T, Girmay G, Lemma M, Negash M. Knowledge, Attitude, and Practice towards Hepatitis B Virus among Pregnant Women Attending Antenatal Care at the University of Gondar Comprehensive Specialized Hospital, Northwest Ethiopia. Int J Hepatol. 2020;2020. doi: 10.1155/2020/5617603 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246915.ref003] 3.Zheng Y, Wu J, Ding C, Xu K, Yang S, Li L. Disease burden of chronic hepatitis B and complications in China from 2006 to 2050: An individual-based modeling study. Virol J. 2020;17: 1–10. doi: 10.1186/s12985-019-1274-x [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246915.ref004] 4.Di Pasquale A, Preiss S, Da Silva FT, Garçon N. Vaccine adjuvants: From 1920 to 2015 and beyond. Vaccines. 2015;3: 320–343. doi: 10.3390/vaccines3020320 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246915.ref005] 5.Djurisic S, Jakobsen JC, Petersen SB, Kenfelt M, Klingenberg SL, Gluud C. Aluminium adjuvants used in vaccines. Cochrane Database Syst Rev. 2018;2018. doi: 10.1002/14651858.CD013086 [DOI] [Google Scholar]

[pone.0246915.ref006] 6.Meireles LC, Marinho RT, Van Damme P. Three decades of hepatitis B control with vaccination. World J Hepatol. 2015;7: 2127–2132. doi: 10.4254/wjh.v7.i18.2127 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246915.ref007] 7.Zimmermann P, Curtis N. Factors That Influence the Immune Response to Vaccination. Am Soc Microbiol. 2019;32: 1–50. doi: 10.1128/CMR.00084-18 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246915.ref008] 8.Arhewoh MI, Eraga SO, Builders PF, Ibobiri MA. Effect of mucin extraction method on some properties of metronidazole mucoadhesive loaded patches. J Pharm Bioresour. 2015;12: 1–7. [Google Scholar]

[pone.0246915.ref009] 9.Harti AS, Sulisetyawati SD, Murharyati A, Oktariani M. The effectiveness of snail slime and chitosan in wound healing. Int J Pharma Med Biol Sci. 2016;91: 2–6. [Google Scholar]

[pone.0246915.ref010] 10.Momoh MA, Chime SA, Ogbodo DU, Akudike PK, Udochukwu SU, Ossai EC, et al. Biochemical, rheological and hydrophile-lipophile balance (HLB) evaluation of archachatina marginata (snail) mucin extract for possible nutraceutical and nano biopharmaceutical applications. Trop J Pharm Res. 2019;18: 927–934. doi: 10.4314/tjpr.v18i5.3 [DOI] [Google Scholar]

[pone.0246915.ref011] 11.Kalu AA, Ogbonnaya E, Rabiu KM, Uche EM, Zabaratu Y. Effect of Archachatina marginata mucin on the aggressive factors of gastric ulcer challenged wistar rat stomach tissue. GSC Biological and Pharmaceutical Sciences. 2019;9(1): 077–082. doi: 10.30574/gscbps.2019.9.1.0163 [DOI] [Google Scholar]

[pone.0246915.ref012] 12.Adikwu MU, Nnamani PO. Some physiological and toxicological properties of snail mucin. Bioresearch. 2005;5: 1–6. [Google Scholar]

[pone.0246915.ref013] 13.Carr JH, Rodak BF. Clinical Hematology Atlas 3rd Edition. Saunders: Elsevier. 2009. [Google Scholar]

[pone.0246915.ref014] 14.Thomas L. Alanine aminotransferase (ALT), Aspartate aminotransferase (AST). in Clinical Laboratory Diagnostics. 1st ed. Thomas L, editor. Frankfurt: TH-Books Verlagsgesellschaft; 1998. [Google Scholar]

[pone.0246915.ref015] 15.Moss DW, Henderson AR. Clinical enzymology. 3rd ed. Burtis CA, Ashwood ER, editors. Philadelphia: Tietz Textbook of Clinical Chemistry; 1999. [Google Scholar]

[pone.0246915.ref016] 16.Nejati S, Mirzaee S. N, Nouri HR, Farhoudi R, Namvar NA, Yazdi MH, et al. Immune responses of mice immunized with HBsAg formulated in naloxone/alum mixture: comparison to Fendrix vaccine. Hepat Mon. 2017;17: 44536. [Google Scholar]

[pone.0246915.ref017] 17.O’Leary NA, Wright MW, Brister JR, Ciufo S, Haddad D, McVeigh R. et al. Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional annotation. Nucleic Acids Res. 2016;44: D733–745. doi: 10.1093/nar/gkv1189 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246915.ref018] 18.Launay G, Simonson T. Homology modelling of protein-protein complexes: A simple method and its possibilities and limitations. BMC Bioinformatics. 2008;9: 1–16. doi: 10.1186/1471-2105-9-1 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246915.ref019] 19.Waterhouse A., Bertoni M, Bienert S, Studer G, Tauriello G, Gumienny R, et al. SWISS-MODEL: homology modelling of protein structures and complexes homology modelling of protein structures and complexes. Nucleic Acids Res. 2018;46: W296–W300. doi: 10.1093/nar/gky427 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246915.ref020] 20.Li D, He W, Liu X, Zheng S, Qi Y, Li H, et al. A potent human neutralizing antibody Fc-dependently reduces established HBV infections. Elife. 2017;6: e26738. doi: 10.7554/eLife.26738 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246915.ref021] 21.Schijns V, Fernández-Tejada A, Barjaktarović Ž, Bouzalas I, Brimnes J, Chernysh S, et al. Modulation of immune responses using adjuvants to facilitate therapeutic vaccination. Immunol Rev. 2020;296: 169–190. doi: 10.1111/imr.12889 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246915.ref022] 22.Lazic SE, Semenova E, Williams DP. Determining organ weight toxicity with Bayesian causal models: Improving on the analysis of relative organ weights. Sci Rep. 2020;10: 1–12. doi: 10.1038/s41598-019-56847-4 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246915.ref023] 23.Yuan L, Wang Y, Ma X, Cui X, Lu M, Guan R, et al. Sunflower seed oil combined with ginseng stem-leaf saponins as an adjuvant to enhance the immune response elicited by Newcastle disease vaccine in chickens. Vaccine. 2020;38: 5343–5354. doi: 10.1016/j.vaccine.2020.05.063 [DOI] [PubMed] [Google Scholar]

[pone.0246915.ref024] 24.McGill MR. The past and present of serum aminotransferases and the future of liver injury biomarkers. EXCLI J. 2016;15: 817–828. doi: 10.17179/excli2016-800 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246915.ref025] 25.Shibabaw T, Dessie G, Molla MD, Zerihun MF, Ayelign B. Assessment of liver marker enzymes and its association with type 2 diabetes mellitus in Northwest Ethiopia. BMC Res Notes. 2019;12: 1–5. doi: 10.1186/s13104-018-4038-6 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246915.ref026] 26.Nanishi E, Dowling DJ, Levy O. Toward precision adjuvants: Optimizing science and safety. Curr Opin Pediatr. 2020;32: 125–138. doi: 10.1097/MOP.0000000000000868 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246915.ref027] 27.Iwasaki A, Medzhitov R. Control of adaptive immunity by the innate immune system. Nat Immunol. 2015;16: 343–353. doi: 10.1038/ni.3123 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246915.ref028] 28.O’Connell KE, Mikkola AM, Stepanek AM, Vernet A, Hall CD, Sun CC, et al. Practical murine hematopathology: A comparative review and implications for research. Comp Med. 2015;65: 96–113. [PMC free article] [PubMed] [Google Scholar]

[pone.0246915.ref029] 29.Selders GS, Fetz AE, Radic MZ, Bowlin GL. An overview of the role of neutrophils in innate immunity, inflammation and host-biomaterial integration. Regen Biomater. 2017;4: 55–68. doi: 10.1093/rb/rbw041 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246915.ref030] 30.Teng TS, Ji AL, Ji XY, Li YZ. Neutrophils and immunity: From bactericidal action to being conquered. J Immunol Res. 2017;2017. doi: 10.1155/2017/9671604 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246915.ref031] 31.Irvine DJ, Aung A, Silva M. Controlling timing and location in vaccines Darrell. Adv Drug Deliv Rev. 2020;158: 91–115. doi: 10.1016/j.addr.2020.06.019 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246915.ref032] 32.Vono M, Lin A, Norrby-Teglund A, Koup RA, Liang F, Loré K. Neutrophils acquire the capacity for antigen presentation to memory CD4+ T cells in vitro and ex vivo. Blood. 2017;129: 1991–2001. doi: 10.1182/blood-2016-10-744441 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246915.ref033] 33.Provencher BA, Everds NE, Zimmerman KL, Moore DM, Smith SA, Barnhart KF. Hematology of laboratory animals. 1st ed. Weiss D, Wardrop J, Chalm OW, editors. Hoboken (NJ): Wiley-Blackwell: Schalm’s Veterinary Hematology; 2010. doi: 10.1258/la.2010.009049 [DOI] [Google Scholar]

[pone.0246915.ref034] 34.Lin C-Y, Lin S-J, Yang Y-C, Wang D-Y, Cheng H-F, Yeh M-K, et al. Human Vaccines & Immunotherapeutics Biodegradable polymeric microsphere-based vaccines and their applications in infectious diseases Biodegradable polymeric microsphere-based vaccines and their applications in infectious diseases. 2015;11: 650–656. Available: http://www.tandfonline.com/action/journalInformation?journalCode=khvi20%5Cn 10.1080/21645515.2015.1009345. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246915.ref035] 35.Joshi VB, Geary SM, Salem AK. Biodegradable particles as vaccine antigen delivery systems for stimulating cellular immune responses. Hum Vaccines Immunother. 2013;912: 2584–2590. doi: 10.4161/hv.26136 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246915.ref036] 36.Gutjahr A, Phelip C, Coolen AL, Monge C, Boisgard AS, Paul S, et al. Biodegradable polymeric nano particles-based vaccine adjuvants for lymph nodes targeting. Vaccines. 2016;4: 34. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246915.ref037] 37.Kondo Y, Ninomiya M, Kakazu E, Kimura O, Shimosegawa T. Hepatitis B Surface Antigen Could Contribute to the Immunopathogenesis of Hepatitis B Virus Infection. ISRN Gastroenterol. 2013;2013: 1–8. doi: 10.1155/2013/935295 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246915.ref038] 38.Shinde P, Howie HL, Stegmann TC, Hay AM, Waterman HR, Szittner Z, et al. IgG Subclass Determines Suppression Versus Enhancement of Humoral Alloimmunity to Kell RBC Antigens in Mice. Front Immunol. 2020;11: 1516. doi: 10.3389/fimmu.2020.01516 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0246915.ref039] 39.Chiu ML, Goulet DR, Teplyakov A, Gilliland GL. Antibody Structure and Function: The Basis for Engineering Therapeutics. Antibodies. 2019;8: 55. doi: 10.3390/antib8040055 [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

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

Parker Elijah Joshua

Cynthia Ogochukwu Nwauzor

Damian Chukwu Odimegwu

Uzochukwu Gospel Ukachukwu

Rita Onyekachukwu Asomadu

Timothy Prince Chidike Ezeorba

Roles

Abstract

Introduction

Materials and methods

Animals

Reagents

Vaccine and secondary antibodies

Snails

Extraction of snail mucin

Preparation of snail mucin dispersion (adjuvant)

Experimental design

Immunization protocol

Determination of body weights and liver weight

Differential white blood cell count

Liver function test of mice immunized with snail mucin-adjuvanted rHBsAg vaccine

Measurement of antibody titres of IgG, IgG1 and IgG2a against rHBsAg using indirect Enzyme-Linked Immunosorbent Assay (iELISA)

Statistical analysis

Homology modelling of mucus protein and model validation

Homology modelling of Hepatitis B surface antigen (HBsAg) and model validation

Protein-protein alignment of snail mucus protein and HBsAg and generating structural morphs with PyMOL 2.4 version 2.4.1

Studies of antigen (HBsAg)–antibody interaction using molecular docking

Results

Snail mucin-adjuvanted rHBsAg vaccine had no adverse effects on experimental mice

Fig 1. Effect of snail mucin-adjuvanted rHBsAg vaccine on the body and liver weight of experimental mice.

Snail mucin-adjuvanted rHBsAg vaccine had no significant toxicity in the liver

Table 1. Effect of snail mucin-adjuvanted rHBsAg vaccine on liver marker enzyme activities of experimental mice.

Snail mucin-adjuvanted rHBsAg vaccine had no significant effect on the granulocyte count of experimental mice

Fig 2. Effect of snail mucin-adjuvanted rHBsAg vaccine on neutrophil count of experimental mice.

Effect of snail mucin-adjuvanted rHBsAg vaccine on cellular immune response (lymphocyte count) of experimental mice

Fig 3. Effect of snail mucin-adjuvanted rHBsAg vaccine on lymphocyte count of experimental mice.

Effect of snail mucin-adjuvanted rHBsAg vaccine on the titre value of immunoglobulin G (IgG), immunoglobulin G subclass 1 (IgG1) and immunoglobulin G subclass 2a (IgG2a) of experimental mice

Fig 4. Effect of snail mucin-adjuvanted rHBsAg vaccine on IgG, IgG1 and IgG2a titre of experimental mice.

Homology modelling of snail mucus protein and HBV envelope protein

Fig 5. Homology modelled structure of snail mucus protein [Cornu aspersum] with accession number QEG59313.1.

Fig 6.

Rheomorphic structural state from interaction of snail mucus protein and modelled HBsAg did not give a better docking score than the standalone HBsAg

Fig 7. Docking morph originating from the interaction of the HBsAg and snail mucus protein with the human immunoglobulin against HBV (PDB_ID– 5YAX) and comparison with the result when standalone HBsAg is docked to 5YAX.

Discussion

Conclusion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Isabelle Chemin

Roles

Author response to Decision Letter 0

Decision Letter 1

Isabelle Chemin

Roles

Author response to Decision Letter 1

Decision Letter 2

Isabelle Chemin

Roles

Acceptance letter

Isabelle Chemin

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases