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For Review OnlyPotential of Chlorella sp. exopolysaccharide as adjuvant for
Mannheimia haemolytica A2 vaccine in rat model.
Journal: Songklanakarin Journal of Science and Technology
Manuscript ID SJST-2019-0494.R1
Manuscript Type: Original Article
Date Submitted by the Author: 20-Apr-2020
Complete List of Authors: Kamaradin, Insyirah-husna; Universiti Malaysia Terengganu, Institute of Marine BiotechnologyAssaw, Suvik; Universiti Malaysia Terengganu, Faculty of Marine Science and Environment; Universiti Sains Malaysia, Department of biomedical science, School of Health ScienceSheikh, Hassan; Universiti Malaysia Terengganu, Faculty of fisheries and food ScienceAbdul Wahid, Mohd Effendy; Universiti Malaysia Terengganu, Faculty of Fisheries and Food Science; Universiti Malaysia Terengganu, Institute of Marine Biotechnology
Keyword: Exopolysaccharide, Mannheimia haemolytica A2, adjuvant, immunoglobulins, intranasal.
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Songklanakarin Journal of Science and Technology SJST-2019-0494.R1 Sheikh
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Manuscript ID SJST-2019-0494 entitled "Potential of Chlorella sp. exopolysaccharide as adjuvant for Mannheimia haemolytica A2 vaccine in rat model."
Comment Response
The degree of increased/decreased (how many times) for each parameters measured are describe in the result part.
We appreciate this comment, however, the increases were significant but not as high as 2 or 3 folds. Here are some examples:
1- IgM titers in EPS-MhA2 treated group starting at week 5 till week 6 increase significantly (about 20%) but wasn’t as high as 2 folds for example.
2- EPS-MhA2 vaccine also significantly increased IgA titer after booster dose at week 5 (about 67%), which is also less than 2 folds.
The levels of the antibodies were expressed as the absorbance unit (Fig 7). Is it possible to show the antibody level in concentration unit? Since the first antibody normally produced during a primary immune response is Ig M. However, the absorbance of Ig G were higher than IgM during wk 0 and wk 1 in both MHA2 and EPS-MHA2 groups.
We didn’t convert the units because first, the data were sufficient to show the effect of vaccination. Secondly, we will need large number of animals to establish a cut-off point which is not practical at this point of the research.
The starting point of IgG was already higher than IgM, because of 2 main reasons. First, IgG is in the serum, while IgA circulates and enter the bloodstream with the secretion of localized BALT in the lungs. Secondly, the animals used are not specific-pathogen free (SPF) animals.
Gough, N. R. (2006) reported that antibody-producing cells undergo a process of differentiation and class switch recombination (CSR) such that the antibodies produced start as immunoglobulin M (IgM) and then switch to IgG and IgA as the concentration of antigen changes and as the cells differentiate. Therefore, the initial antibody levels circulating in the serum were not significantly important since they are not specific to the antigen introduced during the first exposure, with the a condition that it does not exceed twice of the control animal antibody’s level.
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At wk 1 post vaccination (Fig 7), all groups showed gradual increase in Ig A (p > 0.05), except PBS ?? (the original text was MHA2
The correct word is “PBS” and the text has been edited.
The role of Ig A for local immunity should be of concern. Would it be possible to address whether Ig A can be detected in the secretion of upper respiratory tract region ?
Even though the upper respiratory tract is suitable specially to study NALT (nasal-associated respiratory tract), however, the selected pathogen usually colonizes and invade the lower respiratory tract (bronchioles and lungs). In the nasal there is air turbinates that would be difficult to process for histology examination.
Figures : Minor editing on figure legends Figures number corrected
Reference:
Gough, N. R. (2006). How to Get from IgM to IgG. Science Signaling, 2006(358). doi: 10.1126/stke.3582006tw365.
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1 Title: Potential of Chlorella sp. exopolysaccharide as adjuvant for Mannheimia
2 haemolytica A2 vaccine in rat model.
3
4 Insyirah-husna Kamaradin3, Suvik Assaw2,3, Hassan, I. Sheikh1 and Mohd Effendy
5 Abd Wahid1,3*
6
7 1Faculty of Fisheries and Food Science, Universiti Malaysia Terengganu, Terengganu,
8 Malaysia;
9 2Faculty of Marine Science and Environment, Universiti Malaysia Terengganu,
10 Terengganu, Malaysia
11 3Institute of Marine Biotechnology, Universiti Malaysia Terengganu, Terengganu,
12 Malaysia;
13 4Department of Biomedical Sciences, School of Health Science, Unversiti Sains
14 Malaysia, 16150 Kota Bharu, Kelantan.
15
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17 *Corresponding Author: [email protected]
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31 ABSTRACT
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32 The potential of exopolysaccharides (EPS) extracted from Chlorella sp. as adjuvant for
33 formalin-killed whole organism of Mannheimia haemolytica A2 (MhA2) vaccine was
34 investigated. Chlorella sp. was cultured in F/2 media and EPS was extracted and
35 characterized using GPC, FTIR and SEM, while its cytotoxicity was tested on RAW
36 264.7. Sprague Dawley rats were inoculated intranasally with either phosphate-buffered
37 saline (PBS) (Group A), EPS only (Group B), formalin killed whole organism of MhA2
38 vaccine seed only (Group C) or EPS-MhA2 adjuvanted vaccine (Group D). Serum levels
39 of IgM, IgG, and IgA titers were collected for nine weeks. About 4.8 mg of EPS was
40 obtained in the form of white powder with molecular weight of 2398 Da. The
41 microstructure of EPS was compact and exhibited flakes like structural units and was not
42 toxic to RAW 264.7. EPS-MhA2 group produced significantly higher (p<0.05) IgM, IgG
43 and IgA responses compared to other groups.
44
45 Keywords: Exopolysaccharide, Mannheimia haemolytica A2, adjuvant,
46 immunoglobulins, intranasal.
47
48 INTRODUCTION
49 Vaccination is the most effective strategy in the prevention of infectious diseases by
50 generating immune responses and maintain memory cells that able to induce rapid recall
51 responses (Bartlett & Tyring, 2009; Sarkander, Hojyo, & Tokoyoda, 2016). The entry
52 point of more than 90% of pathogens is via mucosal membrane and mucosal vaccination
53 is an effective approach (Miquel‐Clopés, Bentley, Stewart, & Carding, 2019). The
54 vascularized mucosal surface area is 150 cm3 at the nasopharyngeal compartment and can
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55 be targeted for vaccine uptake (Holmgren & Czerkinsky, 2005; Olszewska & Steward,
56 2001; Wang, Liu, Zhang, & Qian, 2015).
57 Pneumonic mannheimiosis caused by Mannheimia haemolytica serotype A2, a gram-
58 negative commensal bacterium that can be found at the upper respiratory tract of domestic
59 and wild animals including healthy ruminants (Roier et al., 2013). The bacterium can
60 turn opportunistic when the animals are exposed to predisposing factors such as
61 pregnancy, transportation or introduction of new animals into an existing herd.
62 Pneumonic mannheimiosis can lead to high mortality and economic losses to the ruminant
63 industry (Jesse et al., 2014).
64 M. haemolytica initiates their infection at the lining mucosal surfaces of the lower
65 respiratory system (Wang et al., 2015). The efficacy of mucosal vaccination through an
66 intranasal route of delivery had been successful in small ruminants (Effendy, Zamri-Saad,
67 Puspa, & Rosiah, 1998a; Effendy, Zamri-Saad, Mohamad, & Omar, 1998b; Zamri-Saad,
68 Effendy, Israf, & Azmi, 1999a; Zamri-Saad, Maswati, Effendy, & Jasni, 1999b).
69 However, the uptake of vaccines from the nasal cavity is poor due to rapid clearance and
70 poor transportation across the nasal mucosal membrane (Svindland et al., 2012).
71 Therefore, the intranasal administration of formalin-killed whole organism of M.
72 haemolytica A2 (MhA2) vaccine would need an extra component, such as an adjuvant
73 that is capable of sustaining their presence and ameliorating the immune responses.
74 Exopolysaccharide (EPS) is an organic macromolecule that consist of various types of
75 sugars and is produced by many microorganisms. The EPS can be found either as capsular
76 matrix that bound covalently to the cell surface or as slime materials that bound loosely
77 (Ahmed, Wang, Anjum, Ahmad, & Khan, 2013; Badel, Bernardi, & Michaud, 2011). In
78 marine environment, EPS form biofilm, which allows the cells to adhere to other bacteria,
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79 animal, plant tissues or inert surfaces (Sutherland, 2001). Due to these properties, EPS
80 could have muco-adhesives character that might be suitable as mucosal adjuvant that
81 could ensure long retention and high absorption rates in the nasal cavity (Svindland et al.,
82 2012; Zaman, Chandrudu, & Toth, 2013).
83 In recent studies, EPS from marine origin such as microalgae has been used in
84 pharmaceuticals industry due to its immune-modulatory, apoptotic and antiviral activity
85 (Xiao & Zheng, 2016; Ishiguro et al., 2017). Hence, EPS from Chlorella sp. was chosen
86 to elucidate its potential as a mucosal adjuvant vaccine for formalin-killed whole
87 organism of Mannheimia haemolytica A2 vaccine. The study aims at evaluating the in
88 vivo adjuvant activity of EPS using Sprague Dawley rat model to determine whether EPS
89 can enhance the humoral immunity in systemic compartments by examining the
90 development of antibodies (IgM, IgG and IgA) responses.
91
92 MATERIALS AND METHOD
93 Extraction of EPS from Chlorella sp.
94 A pure culture of Chlorella sp. (1x106 CFU/mL) that was cultured into F/2 media at ratio
95 of 1:1, was centrifuged at 15,000 rpm for 20 minutes at 4oC using refrigerated centrifuge
96 in order to extract EPS following previously established method with slight modifications
97 (Bajpai, Majumder, Rather, & Kim, 2016). Then, supernatant produced were mixed with
98 100% ice-cold ethanol at ratio 1:1 (25 mL/ 25 mL) and incubated at 4oC overnight. After
99 24 hours of incubation, the solution was centrifuged again at 15,000 rpm for 20 minutes
100 at 4oC. The pellet was collected and washed twice with distilled water then dissolved in
101 distilled water. The pellet was then dialyzed using 16.5 x 26 mm visking tubing
102 membrane for 24 hours at 4oC to remove protein residues. Next, the solution in visking
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103 tube was transferred into the falcon tube and freeze-dried using vacuum freeze dryer. The
104 dried pellet obtained was weighted and stored refrigerated at -20oC.
105 Identification of EPS from Chlorella sp.
106 Gel Permeation Chromatography (GPC)
107 The molecular weight of EPS pellet was analyzed using Gel Permeation Chromatography
108 (GPC) method using a Waters 1515 HPLC system equipped with Waters 2414 refractive
109 index detector (RID). Three types of columns, namely 7.8 x 300 mm UltrahydrogelTM
110 500 columns, 7.8 x 300 mm UltrahydrogelTM 120 columns and 7.8 x 300 mm
111 UltrahydrogelTM Linear columns were used. Then, 1 mg of EPS was dissolved in 1mL of
112 0.1 M NaNO3 and injected. The eluent was 0.1 M NaNO3 in water. The columns and
113 detector were maintained at 35oC. The sample was analyzed within 40 minutes at a flow
114 rate of 1 mL/min and the injection volume was set at 25 μL. Dextran Molecular Weight
115 Standard Kits (Polymers Standards Service-USA, Inc. Germany) was used in the GPC as
116 a standard. The data collection and analysis were carried out using a Breeze 2 software
117 (Xing et al., 2017).
118
119 Fourier Transform Infrared spectroscopy (FTIR)
120 The spectra of EPS were recorded using spectrophotometer (Perkin Elmer, USA) using
121 Potassium Bromide (KBr) disc. FTIR absorption band were read at wavelengths (λ)
122 between 2.5 μm to 25 μm (1 μm = 10-6 m) and measured in cm-1 unit (Siti-Aisha, 2018).
123
124 Scanning Electron Microscope (SEM)
125 The diluted EPS sample (106 CFU/mL) was centrifuged at 4,000 rpm for 15 minutes. The
126 pellet obtained was placed on a small circle glass slide and dried in room temperature for
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127 15 minutes. Next, the slide with dried pellet was placed on stubs, then coated with the
128 gold 20-40 μm thicknesses using auto fine coated JFC 1600 and viewed under SEM (Akin
129 & Amos, 1975).
130
131 Cytotoxicity test of EPS
132 Measurement of cell viability assay by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-Diphenyl-
133 Tetrazolium Bromide (MTT)
134 Viability of the cell against EPS was determined via 3-(4,5-dimethylthiazol-2-yl)-2, 5-
135 Diphenyl-Tetrazolium Bromide (MTT) assay (Razali et al., 2014). Briefly, RAW 264.7
136 cells that obtained from the American Type Culture Collection (ATCC) was cultured
137 according to the method described by Kim et al. (2015). After the cells culture reached
138 80% confluence in the culture flask, cells were seeded in 96 wells plate at the density of
139 5 x 104 cells/well. After 24 hours of incubation, the adhered cells were treated with various
140 concentrations of the EPS that was diluted in DMEM (100 µg/mL, 50 µg/mL, 25 µg/mL,
141 12.5 µg/mL, 6.25 µg/mL, 3.12 µg/mL, and 1.56 µg/mL, Control positive (DMEM only).
142 Twenty-four hours later, MTT was added and the cells were incubated for 4 hours at 37°C
143 and 5% CO2. The medium was then removed, and the formazan precipitate was
144 solubilized in DMSO. The absorbance was measured at 550 nm by using microplate
145 reader (Thermo-Scientific Multiskan AscentTM, USA) for cell viability calculation. The
146 cytotoxicity of EPS was expressed as the IC50, which is the concentration of each EPS
147 that reduces the 50% of cell survival when compared with the control.
148 Cell viability (%) = (OD550 of sample) (OD550 of control)-1 x 100
149
150
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151 Preparation of formalin-killed cells (FKCs) of Mannheimia haemolytica A2 (MhA2)
152 vaccine
153 Mannheimia haemolytica A2 were cultured on Thermo Scientific Columbia Sheep Blood
154 Agar (BA) plate. About 30 colonies of the pure bacteria were transferred into 200 mL of
155 Brain Heart Infusion broth. The culture was then shook by using shaker incubator at 37°C
156 for 18 hours for replications. Following incubation, serial dilutions and standard plate
157 counting were done to determine the concentration of the culture. The bacteria were then
158 killed by introducing neutral-buffered formalin at the concentration of 1% (0.25 mL/ 25
159 mL) formalin in phosphate buffered saline and kept overnight at 4°C. Then the bacteria
160 were washed five times with PBS by centrifugation at 6000 xg for 15 minutes using
161 refrigerated centrifuge to eliminate the remaining formalin from the cultures. The bacteria
162 were washed again five times with sterile PBS, and the concentration in preparation was
163 adjusted to 106 CFU/mL using optical density (Noraini, Sabri, & Siti-Zahrah., 2013).
164
165 Preparation of exopolysaccharide-Mannheimia haemolytica A2 (EPS-MhA2)
166 vaccine
167 The 4.8 mg of EPS powder were diluted in 400 mL of phosphate buffered saline to prepare
168 as a stock solution with a concentration 0.012 mg/mL. Then only 30 μL of diluted EPS
169 was taken and mixed together with the pellet of 106 CFU/mL of formalin-killed M.
170 haemolytica A2 (MhA2) vaccine to make exopolysaccharide-M. haemolytica A2 (EPS-
171 MhA2) vaccine (Noor-Hidayah, 2014).
172
173
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174 Confirmation of exopolysaccharide-Mannheimia haemolytica A2 (EPS-MhA2)
175 vaccine
176 Scanning Electron Microscope (SEM)
177 The mixture of diluted EPS and 106 CFU/mL of MhA2 vaccines were centrifuged at 4,000
178 rpm for 15 minutes using centrifuge. Then, the pellet was collected and placed on small
179 round glass slide and dried in room temperature for 15 minutes. The slide with dried pellet
180 was coated with the gold 20-40 μm thicknesses using auto fine coated JFC 1600 and ready
181 to view in low vacuum condition under SEM (Akin and Amos, 1975).
182
183 Experimental design of white rats (Sprague Dawley)
184 Twelve female healthy Sprague Dawley of 6-8 weeks age were selected and allocated
185 randomly into 4 groups with 3 rats each group assigned as group A, group B, group C
186 and group D. Group A and B were negative control treated with phosphate buffered saline
187 and EPS as adjuvant respectively. Group C was positive control treated with MhA2
188 vaccine only and Group D was treated with EPS-MhA2 vaccine, which served as
189 adjuvanted vaccine. All Sprague Dawley were inoculated by dripping 30 µL of samples
190 via the intranasal route. Blood samples were taken every week from each of the Sprague
191 Dawley’s tail vein for determining the antibody production of the rats. The first treatments
192 were administered into the Sprague Dawley’s nostril and labeled as week 0. Then on week
193 2, second booster dose was given. Six weeks after 2nd vaccination (week 8), all rats were
194 euthanized according to the animal ethics code UMT/JKEPHT/2017/2.
195
196
197
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198 RESULTS
199 Physical and chemical characterization of exopolysaccharide (EPS) from Chlorella
200 sp.
201 The 400 mL of Chlorella sp. culture containing 106 CFU/mL had yielded 4.8 mg in the
202 form of white powder that and was readily soluble in water (Figure 1). Gel Permeation
203 Chromatography (GPC) revealed that the average molecular weight (Mw) of the EPS was
204 2398 Da as shown in (Figure 2). FTIR spectrum indicates the major functional groups
205 and chemical bonds present in EPS. The peak at 3350.35cm-1 indicated O-H stretch. A
206 weak absorption band at the wave-number region <3000 cm-1 was assigned to the
207 symmetric stretching vibration of CH group. A sharp peak found at 1683.86 cm-1 and
208 1402.25 cm-1 indicated stretching vibrations of C=O and C-O respectively which are
209 linked to COOH groups. While the absorption bands in the region 1116.78 cm-1 and
210 975.98cm-1 suggested a typical signature of carbohydrates molecules (Figure 3).
211 Morphology of freeze-dried EPS was visualized by Scanning Electron microscope (SEM)
212 analysis at 2500x magnification. The microstructure of freeze-dried EPS was found to
213 have compact in structure and flakes like structural unit structure with sharp edges (Figure
214 4).
215
216 Cytotoxicity of exopolysaccharide (EPS) from Chlorella sp.
217 The in vitro cytotoxicity test of EPS was evaluated using MTT assay on Mouse
218 Leukaemic monocyte-macrophages cell line (RAW 264.7). The MTT assay was
219 performed as percentage relative viability of cells (%) versus concentrations of EPS
220 extracts (100 µg/mL, 50 µg/mL, 25 µg/mL, 12.5 µg/mL, 6.25 µg/mL, 3.12 µg/mL, and
221 1.56 µg/mL) and the percentage viability of RAW 264.7 cell was calculated after 24 hours
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222 exposure in concentration-dependent manner. The results as shown in Figure 5 indicated
223 that EPS promoted the growth of RAW 264.7 cell at all concentrations (3.125 µg/mL
224 (112.21%), 6.25 µg/mL (125.07%), 12.5 µg/mL (140.34%), 25 µg/mL (143.97%), 50
225 µg/mL (152.01%), 100 µg/mL (140.84%)) except at this concentrations 1.56 µg/mL
226 (67.24%) of EPS where the RAW 264.7 growth was lower than control (100%). There
227 was no significant differences (p>0.05) in percentage relative viability of cell between
228 tested concentrations and the control. No IC50 value was obtained, hence, EPS is safe at
229 all tested concentrations.
230
231 Morphology of exopolysaccharide-Mannheimia haemolytica A2 (EPS-MhA2)
232 vaccine
233 In order to confirm whether the EPS powder able to encapsulate whole-cell formalin
234 killed M. haemolytica A2, the morphological structure of the EPS-MhA2 was observed
235 under SEM. Based on Figure 6, the clumping structure showed the EPS had successfully
236 encapsulated the whole-cell formalin-killed M. haemolytica A2 under 6,1500x
237 magnification.
238
239 Development pattern of IgM, IgA and IgG of white rats (Sprague Dawley)
240 Enzyme-linked Immunosorbent Assay (ELISA) results revealed that there were three
241 types of response patterns showed by IgM, IgG and IgA antibodies in the blood serum of
242 experimental Sprague Dawley rats before and after vaccination (Figure 7). At the starting
243 week (week 0) before vaccination program, the levels of IgM, IgG and IgA in all Sprague
244 Dawley were at low with no significant (p>0.05) differences between groups. However,
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245 following the 1st intranasal vaccination on week 1, the development of IgM, IgG and IgA
246 responses in all groups of Sprague Dawley showed a different pattern of responses.
247 For the IgM, after the 1st intranasal vaccination on week 1, the level of IgM in all groups
248 showed a gradual increase but the increment was not significant (p>0.05) until week 3.
249 However, following a booster dose on week 2, despite slight decrease at week 3, both
250 EPS-MhA2 and M. MhA2 vaccinated groups showed significant (p<0.05) increase at
251 week 4 when compared with both unvaccinated groups. At week 5 and week 6, only EPS-
252 MhA2 group sustained a significant increase (p<0.05) when compared to other groups.
253 For IgG, at week-1 post intranasal vaccination, the mean antibody levels of IgG showed
254 significant (p<0.05) increase in EPS-MhA2 and M. MhA2, while insignificant (p>0.05)
255 gradual increase was observed in EPS-only and PBS groups before the mean antibody
256 level started to decline at week 2 and 3 post-vaccination. After that, the 2nd intranasal
257 vaccination of EPS-MhA2 and MhA2 vaccine group showed that it had successfully
258 induced IgG production. However, EPS-MhA2 vaccine group produced highest and
259 significant (p<0.05) increase when compared with MhA2 vaccine group and the
260 unvaccinated control groups at week 5. Meanwhile for IgA, similar pattern with IgG was
261 observed. At week 1 post intranasal vaccination all groups except PBS showed gradual
262 increase in antibody, but the levels were not significant (p>0.05). The booster dose
263 showed again that EPS-MhA2 and MhA2 vaccine group significantly (p<0.05) increased
264 the antibody levels of IgA at week 5 as compared to the control groups. Among
265 vaccinated groups, IgA in EPS-MhA2 vaccine group was still significantly (p<0.05)
266 higher than MhA2 vaccine group.
267 Overall, from the development pattern of IgM, IgG and IgA, only EPS-MhA2 group
268 consistently demonstrated a significant increased (p<0.05) in the production of IgM, IgG
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269 and IgA, especially after administration of booster doses at two weeks interval. The level
270 of IgM started to increase after the 2nd booster on week 4 and peaked at week 6, while
271 IgG and IgA reach their peak at week 5, respectively when compared with the rest of the
272 non-adjuvanted groups.
273
274 DISCUSSION
275 Chlorella sp. is known to produce and excrete large amounts of EPS in their extracellular
276 environment (Delattre, Pierre, Laroche, & Michaud, 2016). Chlorella sp. culture medium
277 was used to extract EPS. The yield EPS obtained in this study from Chlorella sp. culture
278 using 106 CFU/mL was about 120 mg/L. In another study, yield of EPS obtained from
279 Chlorella sp. was 174 mg/L (Xiao & Zheng, 2016), however, the study didn’t report the
280 concentration of the Chlorella sp. Physiochemical, structural, and biological activity of
281 the EPS were determined to assess their biodegradability and toxicity to be used as
282 vaccine adjuvant (Ahmed et al., 2013). EPS produced in this study was in the form of
283 white powder and was highly water soluble. The EPS showed a single symmetrical peak
284 in Gel Permeation Chromatography (GPC) profile, which indicated that the extracted EPS
285 was a homogeneous polysaccharide (Du, Yang, Bian, & Xu, 2017). The average
286 molecular weight (Mw) of purified EPS was 2398 Da, which was relatively small in size
287 and categorized as low molecular weight. Xiao and Zheng (2016) suggested that EPS
288 from Chlorella sp. should have high molecular weight due to its structural
289 polysaccharides, which mainly consist of 1-6 galactose linkage. Noor-Hidayah (2014)
290 also reported low molecular weight of Chlorella sp. to be as low as 2274 Da. The low
291 molecular weight EPS could be due to degradation of polysaccharide chain (Ziadi et al.,
292 2018) or due to limited number of monosaccharides present such as glucose, xylose,
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293 glucuronic acid and galactose (Raposo, de Morais, and de Morais, 2015; Xiao & Zheng,
294 2016). However, low molecular weight is advantageous as it associated with higher water
295 solubility, stability and organism absorption (Ramnani et al., 2012), hence, suitable to be
296 used as adjuvant vaccines. Fourier-transform infrared spectroscopy (FTIR) spectra of
297 Chlorella sp. EPS was in agreement with previous studies (Bayona & Garcés, 2014; Sajna
298 et al., 2013; Wang, 2011). The OH group at 3350.35 cm-1 could allow EPS to be absorbed
299 easily by the mucus of nasal cavity and increase the nasal residence time for the vaccine
300 (Sahoo, Chakraborti, & Mishra, 2011; Yang, Zhao, & Fang, 2008; Zaman et al., 2013).
301 SEM properties of Chlorella sp.’s EPS were similar to the properties of EPS reported in
302 previous studies (Ahmed et al., 2013; Du et al., 2017; Piermaria, Pinotti, Garcia, &
303 Abraham, 2009) where the surface of EPS was dull and had pores. The microstructure of
304 the produced EPS was similar to materials used to make plasticized films which indicated
305 high water solubility, water absorbency and biodegradability. The EPS was also
306 homogeneous matrix, which indicated the structural integrity and ability to make a film
307 (Ahmed et al., 2013). The morphology of EPS changed when it was mixed with sterile
308 PBS excipient and the seed of formalin-killed whole organism MhA2 (Figure 6).
309 Variations in EPS structure is common due to differences in physicochemical properties
310 of the polysaccharide itself (Kanamarlapudi & Muddada, 2017). Chlorella sp.’s EPS did
311 not show any cytotoxic effect on RAW 264.7 macrophage cell by MTT assay at all the
312 tested concentrations between 1.56 to 100 µg/ml. This finding is consistent with previous
313 study where the polysaccharide extracted from green microalgae Haematococcus
314 lacustris on RAW 264.7 macrophage cells had immune-modulating activity without any
315 detectable level of cytotoxicity (Park et al., 2011).
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316 Chlorella sp. EPS potential to be used as an adjuvant for M. haemolytica A2 (MhA2)
317 vaccine at mucosal surfaces. The development patterns of the antibody in the blood serum
318 are direct indicator of the humoral immune responses and generally used for examining
319 the effectiveness of vaccine (Yang et al., 2015). In this study, IgM, IgG and IgA
320 production was monitor in the blood serum of Sprague Dawley (Figure 7). Generally,
321 EPS-MhA2 adjuvanted vaccine group showed higher level of antibodies, especially after
322 2nd intranasal vaccination at week 2. Significant increase in IgM titers in EPS-MhA2
323 treated group starting at week 4 till week 6 could be due to opsonizing effect of serum
324 IgM towards the exposure of EPS-MhA2. This result was supported by the fact that the
325 IgM would be secreted out into the blood as the first antibody production to take action
326 during the primary and secondary responses of any antigen (Black & Black, 2008). Then,
327 the antibody response was followed by IgG production. In this study, there was increase
328 of IgG level in Sprague Dawley in the two vaccinated groups (EPS-MhA2 and MhA2).
329 The increase of IgG level from these groups was gradually stimulated starting at week 4
330 and then became significantly high at week 5. However, EPS-MhA2 vaccine showed
331 significantly higher IgG responses compared to MhA2 only vaccine. This observation
332 indicated that the EPS as adjuvant had a good ability to maintain the M. haemolytica A2
333 vaccine in mucosal region of the Sprague Dawley, thus gave appropriate time for this
334 antigens to stimulate memory cells during primary response. Due to the adequate
335 production of these memory cells, the administration of the booster dose invigorated the
336 significantly high level of IgG on systemic immune compartment. This finding was
337 similar to the previous study where EPS adjuvant from Aphanothece halophytica
338 (EPSAH) significantly enhanced serum OVA-specific total IgG and IgG2a production in
339 immunized mice at 2 weeks post-immunization (Lei et al., 2016). EPS-MhA2 vaccine
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340 also significantly increased IgA titer in the blood serum of Sprague Dawley at week 5,
341 especially after booster dose (week 2) in EPS-MhA2 group compared to the other groups
342 before the production of IgA declined on week 6. The increase in IgA production in this
343 study was consistent with the observations of IgA responses on mice that were vaccinated
344 with the chitosan polysaccharide-adjuvanted vaccine (Svindland et al., 2012).
345 The findings from this study suggest that the mechanism of the adjuvant is that the
346 microencapsulation of the seeds will protect the seed from being phagocytized by the
347 cells of the innate immune system. Neutrophils will engulf the adjuvant and degrade them,
348 eventually exposing the seeds. Macrophages will clear the remaining EPS capsules and
349 after a few hours, the process continues with the bindings of B cells receptors with the
350 naked antigen seeds before the proliferation or cloning process of antibodies took place
351 (Sompayrac, 2019). The receptors will bind directly to the antigens and continue the
352 cloning process to produce huge quantities of specific antibodies into the tissues and
353 blood. Instead of two weeks interval, it is better to give the booster at three weeks interval
354 due to get more impactful immune responses due to the delay from the innate immune
355 system to remove the EPS.
356
357 CONCLUSION
358 EPS from Chlorella sp. was found to possess suitable physiochemical properties to be a
359 good adjuvant for intranasal delivery. EPS from Chlorella sp. significantly increase of
360 IgM, IgG and IgA humoral immune responses and hence an effective adjuvant for M.
361 haemolytica A2 vaccine. EPS as intranasal adjuvant enablabled M. haemolytica A2
362 vaccine seed to stay longer on the nasal mucosal epithelium. This will aid antigens to get
363 better access to the lymphoid tissue and thereby stimulate an appropriate immune
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364 response in preventing the establishment of this particular infection. EPS obtained from
365 Chlorella sp. could also be used as an adjuvant in other vaccines, especially those
366 administered through intranasal or mucosal areas.
367
368 ACKNOWLEDGEMENT
369 The study was funded by the Fundamental Research Grant Scheme (FRGS) (VOT
370 59338) from the Ministry of higher Education
371
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Figure number Figure titleFigure 1 The photomicrograph of a) cultured Chlorella sp. used in this
study and b) the physical appearance of EPS extracted from cultured Chlorella sp. as freeze-dried powder. Total magnification = 400x (Scale bar = 50 µm).
Figure 2 The chromatogram molecular weight of EPS extracted from Chlorella sp. using Gel Permeation Chromatography (GPC).
Figure 3 FTIR spectrum of crude EPS extracted from Chlorella sp. showing the typical structure and functional groups
Figure 4 SEM image of freeze-dried powder of EPS extracted from Chlorella vulgaris. Total magnification 2500x (Scale bar = 10 µm).
Figure 5 The graphs showed percentage relative viability of RAW 264.7 macrophage cell that was treated with various concentrations of EPS diluted in DMEM (100 µg/mL, 50 µg/mL, 25 µg/mL, 12.5 µg/mL, 6.25 µg/mL, 3.12 µg/mL and 1.56 µg/mL). The control group was treated with complete DMEM only. Values are mean ± SEM, (n=3); One-way ANOVA was performed followed by Tukey’s multiple comparisons test for analysis.
Figure 6 Figure SEM images of exopolysaccharide (EPS) mixed with sterile PBS as excipient able to capsulate M. haemolytica A2 vaccine seed. Total magnification 6,1500x (Scale bar = 1 µm). White arrow: Fragment of Exopolysaccharide, Black arrow: Mannheimia haemolytica A2 (MhA2) vaccine seed
Figure 7 The pattern of responses of IgM, IgG and IgA in Sprague Dawley for each week of experiment. Values are mean ± SEM, (n=3) rats per group; One-way ANOVA was performed using the Tukey’s multiple comparisons test for analysis.
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2021
2223 Figure 1 The photomicrograph of a) cultured Chlorella sp. used in this study and b) the 24 physical appearance of EPS extracted from cultured Chlorella sp. in freeze-dried powder. 25 Total magnification = 400x (Scale bar = 50 µm).26
a) b)
50 µm
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2728
293031 Figure 2 The chromatogram molecular weight of EPS extracted from Chlorella sp. using 32 Gel Permeation Chromatography (GPC).3334
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3536 Figure 3 FTIR spectrum of crude EPS extracted from Chlorella sp. showing the typical 37 structure and functional groups 38
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394041424344454647484950515253545556 Figure 4 SEM image of freeze-dried powder of EPS extracted from Chlorella vulgaris. 57 Total magnification 2500x (Scale bar = 10 µm).58
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5960 Figure 5 The graphs showed percentage relative viability of RAW 264.7 macrophage cell 61 that was treated with various concentrations of EPS diluted in DMEM (100 µg/mL, 50 62 µg/mL, 25 µg/mL, 12.5 µg/mL, 6.25 µg/mL, 3.12 µg/mL and 1.56 µg/mL). The control 63 group was treated with complete DMEM only. Values are mean ± SEM, (n=3); One-way 64 ANOVA was performed followed by Tukey’s multiple comparisons test for analysis.65
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666768 Figure 6 SEM images of exopolysaccharide (EPS) mixed with sterile PBS as excipient 69 able to capsulate M. haemolytica A2 vaccine seed. Total magnification 6,1500x (Scale 70 bar = 1 µm). White arrow: Fragment of Exopolysaccharide, Black arrow: Mannheimia 71 haemolytica A2 (MhA2) vaccine seed.72
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73747576
77 Figure 7 The development patterns of IgM, IgG and IgA responses in Sprague 78 Dawley for each week of experiment. Note that EPS-MHA2 vaccine: 79 Exopolysaccharide-M. haemolytica A2 vaccine, MHA2 vaccine: M. haemolytica A2 80 vaccine, EPS: Exopolysaccharide, PBS: Phosphate buffered saline. Values are mean ± 81 SEM, (n=3) rats per group; One-way ANOVA was performed followed by Tukey’s 82 multiple comparisons test for analysis.
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