APPLICATION OF JACALIN ISOLATED FROM JACKFRUIT (ARTOCARPUS HETEROPHYLLUS LAMK) SEED AS VIRUS AVIAN INFLUENZA A (H5N1) VACCINE ADJUVANT

• Main Author: Riyani, Ani
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/79456
• Institution: Institut Teknologi Bandung

Increasing immune response by a vaccine immunogen often requires the addition of an adjuvant. One potential adjuvant is a lectin obtained from plants such as jacalin. This lectin is a glycoprotein isolated from jackfruit (Artocarpus heterophyllus Lamk) seed. This hypothesis is based on the ability of jacalin to activate T and B cells and also induce humoral immune responses. Furthermore, specific recognition of jacalin to carbohydrate moieties exist on the surface of various mammalian cells is promising as vehicle of avian influenza A (H5N1) virus, which is designed to bind the hemagglutinin of the virus. The study began with jacalin isolation from jackfruit seeds and further characterization, analysis of jacalin binding to H5Nl, evaluation of the adjuvant activity of jacalin by immunogenicity testing in vivo in BALB / c mice and splenocytes proliferation determination in vitro. Dried jackfruit seeds were extracted with pH 7.2 PBS and jacalin was isolated by affinity chromatography using epichlorohydrin cross-linked guargum. The isolated jacalin was characterized by hemagglutination test, hemagglutination inhibition by eight carbohydrates, and the confirmation of protein fraction using eletroforesis SDSPAGE. Subsequently, jacalin was reacted with inactivated 1--15N1 virus of A/CK/WJ/PWT-Wij/06 variant (J-H5Nl). Jacalin interaction with H5Nl virus was analyzed by alteration of the hemagglutination activity and morphological observation using TEM. In addition, the stability of the J-H5Nl was determined at various pH and temperature. Adjuvant activity ofjacalin in H5Nl vaccine was confirmed by determination of immunogenicity enhancement conducted in BALB/c mice in vivo via intra muscular injection and it was booster after 14 days. The response immune levels were determined from hemagglutination titer (HA) as a marker of humoral immune response and cytokine levels (IFN-Y and IL-12) as an indicator of cellular immune response. Hemagglutination Liters were determined by hemagglutinalion inhibition test and precipitation, and the levels ofIFN-y and IL-12 in the serum by ELISA. The adjuvant activity was also analyzed in vitro by determining splenocyte proliferation using MTT method. The yield ofjacalin isolated was circa 0.32% w/w of the dried jackfruit seeds. The jacalin identity was confirmed using SDS-PAGE electrophoresis that showed two bands of about 14.4 and 17.5 kDa. The same result was also shown by jacalin standard (Biovision). Furthermore, it was also confirmed by hemagglutination inhibition test using eight sugars. The isolated jacalin showed specific binding to galactose at the lowest concentration of 6.25mM. The binding of jacalin and H5N1 (J-H5N1) was shown by reduction of hemagglutination activity of jacalin. It was also confirmed by morphological observation using TEM that showed the interaction or bonding among jacalin and H5N1 virus particles in the composite. The J-H5Nl interaction was stable upon heating upto 500C for 30 minutes and also at the pH range of 4-8 shown by the maintainance of it's hemaggiutination activity (at 256 HA titer). Study of morphology with electron microscope examination (TEM) obtained viruses formed spherical with a size of between 100-120 nm, jacalin nearly circular shape with a size of 50-100 nm and a combination of jacalin with the virus seen as a composite that shows the interaction or bonding between the jacalin with the virus H5N1. In silico chemical interaction give a result that saccharide have highest of affinity on H5, this saccharide have free energy, very negative bound are : GalNAc, Metil a Galactose and Metil Galactose, whose free energy in bounding are respectivelly, -5,3; -5,2 and -5,2 kcal/ mol. Methylation of galactose have high afinity with jacalin, where found by docking saccharide against jacalin. The result show that three types of saccharide whose free energy bound very negative are Methyl-a-Galactose, Methyl-a-Manosa, and GalNAc, where free energy of bound are respectivelly; -6,5; -6,1 ; and -5,7 kcal/mol. The result show that vary of galactose and manosa methylation are predicted have a highest affinity, and methylation of galactose known that is a saccharide which found at H5 stucture. In the immunogenicity test of the three concentrations tested jacalin namely 37.5, 50.0 and 75.0 pg/mL (J37,s-H5Nl, Jso-H5N l, J75-H5N l) titers ofhemagglutination (HA) is obtained in 2048 unit and did different significantly from PBS and H5Nl control. Results of the determination of IFN-y and IL-12 from combination of jacalin with a dose of 37.5; 50.0 and 75.0 gg/mL and virus showed an increase in both cytokines was significantly (p <0.05) after the booster and the highest is jacalin obtained at a concentration of 50.0 gg/mL with IFN-Y before injecting 18. 14 ± 0.66 pg/mL and after injection of 25.93 ± 1.58 pg/mL and IL-12 prior to the injection of 16.78 ± 2.17 pg/mL and after injection of 26.45 ± 5.49 pg/mL. The increase in both cytokines also occurred after the administration of adjuvant alum and H5N1 vaccine commercial, but an increase in iFN-y and IL-12 after the administration of combination with influenza virus A (H5Nl) with adjuvant alum and H5N1 vaccine commercial is not significantly increased. These data indicate that both types jacalin and alum adjuvant and H5N1 vacciiie commercial also has the same potential for immunogenicity ofH5NI were used in this study. With the use of alum, IFN-Y before the injection of 18.60 ± 0.95 pg/mL and after injection of 23.95 ± 4.04 pg/mL and IL-12 prior to the injection of 1635 ± 4.45 pg/mL and after injection of 25.40 ± 3.59 pg/mL. By using the H5N1 vaccine, IFN-y before the injection of i 8.81 ± 1.02 pg/mL and after injection of 23.81 2.38 pg/mL and IL-12 prior Lo Lhe injection of 15.95 ± 1.68 pg/mL and after injection of 25.65 3.02 pg/mL. In addition to the cellular immune response in this study also tested the ability of jacalin as an adjuvant in the proliferation of splenocytes isolated from spleen BALB/c mice and cultured in RPMI 1640 medium and determined by MVP assay. Results showed that the three concentrations of jacalin combined with influenza virus A (H5Nl) can induce significant proliferation of splenocytes with the highest proliferation induced after administration of 5gg/mL, are 187.33±0.58%. Similar results occurred after administration of adjuvant alum combination with the H5Nl virus, which is 182.00 5.00% and commercial H5Nl vaccine, 176.00 ± 14.93%. The conclusion, based on test immunogenicity jacalin used has the same potential with alum and the same immunogenicity of the H5Nl vaccine commercial. Blood profiles were also observed for immunogenicity trials showed that administration of three doses of I-I-SNI vaccine with alum and jacalin with commercial vaccine did not affect the number of erythrocytes, hemoglobin concentration, hematocrit, MCV, MCH, and MCHC. These data indicate that the jacalin used in vaccine does not affect blood profile, but significantly increased the lymphocytes, which increased lymphocyte count was higher in the group given the concentration of 50.0 pg/mL. Measurement spieen index was also monitored with a test vaccine (J37,s-H5NlJso-H5N1, J75-H5Ni, alum-H5N1 and H5N1 vaccine). After administration ofthe test vaccine (J50-H5Nl), spleen index of 0.8684 ± 0.11, an increase of 14.3% against PBS and 15.2% against H5Nl, spleen index at HSNl-alum 0.8893 ± 0,21, increased 17, 0% of the control PBS and 18.0% of the control H5Nl, and H5Nl vaccine commercially 0.8715 ± 0.214 spleen index increased by 14.7% against PBS and 15.6% PBS to H5N1, This result equally with HI titer IFN-Y, IL-12 and proliferation of splenocytes. The conclusion of this study is jacalin potentially as an adjuvant in the H5Nl vaccine, while in combination ofjacalin with avian influenza A (H5Nl) has the same immunogenicity with use of adjuvant alum and H5Nl vaccine commercial.