#TITLE_ALTERNATIVE#

• Main Author: TRITAMA NIM : 30714001, ERMAN
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/26967
• Institution: Institut Teknologi Bandung

Typhoid fever, an acute febrile illness and systemic disease caused by Salmonella <br /> <br /> enterica serovar Typhi (S. enterica ser. Typhi), remain a major health problem in <br /> <br /> many developing countries. Typhoid conjugate vaccine (TCV) is an ideal vaccine <br /> <br /> to control, prevent and eliminate typhoid disease due to its ability to induce T-cell <br /> <br /> dependent immune response. Strategic advisory group of experts on immunization <br /> <br /> (SAGE) recommended the introduction of typhoid conjugate vaccine (TCV) for <br /> <br /> infants and children over 6 months .Currently, licensed TCV in the market, using <br /> <br /> tetanus toxoid (TT) as a carrier protein. However, this vaccine had several <br /> <br /> shortages for instance low yield, poor solubility, and depolymerization during <br /> <br /> storage. The aim of this study is to produce the alternative TCV to overcome the <br /> <br /> limitation of the available TCV in the market by analyzing the using of substitute <br /> <br /> carrier proteins and the impact of adjuvant addition in elicit immune response. S. <br /> <br /> enterica ser. Typhi strain C6524 was used and its ability to agglutinate the anti-Vi <br /> <br /> serum was tested. In addition, the genes involved in Vi polysaccharide expression <br /> <br /> were characterized by polymerase chain reaction (PCR). To produce Vi <br /> <br /> polysaccharide, S. enterica ser. Typhi strain C6524 was cultivated in 20 L <br /> <br /> fermenter with agitation 100 ± 10 rpm, temperature 32 ± 1 ºC for 12 ± 1 hours. <br /> <br /> Then, the cells were inactivated using 37% formaldehyde and agitated at 200 ± 10 <br /> <br /> rpm for 20 minutes. Vi polysaccharide (Vi) was isolated using combination <br /> <br /> filtration and ethanol fractionation and characterized by HPLC and NMR. Three <br /> <br /> proteins namely Diphtheria toxoid (DT), Tetanus toxoid (TT) and Hepatitis B <br /> <br /> surface antigen (HB) were chosen as carrier proteins. The proteins were <br /> <br /> derivatized by using adipic acid dihydrazide (ADH) and conjugated to Vi <br /> <br /> polysaccharide using Robins/Kossazka method and designated as Vi-DT, Vi-TT <br /> <br /> and Vi-HB. To measure the progress of conjugation process, the composition of <br /> <br /> the mixture in terms of the conjugated and unconjugated materials was monitored <br /> <br /> via Size-Exclusion Chromatography on an HPLC platform. The yield were <br /> <br /> measured using high-performance anion exchange chromatography-pulse <br /> <br /> amperometric detector (HPAEC-PAD) and show 55.84 ± 2 ; 28.68 ± 2.46 dan <br /> <br /> 4.44 ± 0.98 % of Vi-DT, Vi-TT and Vi-HB, respectively. The vaccines produced <br /> <br /> by formulated Vi-DT, Vi-TT, and Vi-HB, separately, in either aluminum <br /> <br /> phosphate (AlPO4) or aluminum hydroxide (AlOH) as adjuvants and TCV <br /> <br /> iv <br /> <br /> formulated in phosphate buffer saline were used as controls. In each case, a <br /> <br /> group of Balb/c mice was injected intramuscularly with two doses of the <br /> <br /> formulated vaccine at two-week intervals. The anti-Vi IgG responses were <br /> <br /> monitored by Enzyme-Linked Immunosorbent Assay and the levels of CD4 <br /> <br /> + <br /> <br /> Tcells expressing cytokine were characterized using intracellular cytokine staining. <br /> <br /> After the first administration, the mice vaccinated with Vi-TT (AlPO4), Vi-TT <br /> <br /> (AlOH), Vi-TT (PBS), Vi-DT (AlPO4), Vi-DT (AlOH), Vi-DT (PBS), Vi-HB <br /> <br /> (AlPO4), Vi-HB (AlOH), Vi-HB (PBS) elicit anti-Vi IgG 109.368, 57.557, 37.691, <br /> <br /> 53.002, 63.771, 31.864, 16.124, 5.611, and 5.124 times higher, respectively, than <br /> <br /> the group of mice received Vi polysaccharide only. After the second <br /> <br /> administration, All TCV showed giving booster except for Vi-TT (PBS). The <br /> <br /> greatest booster effect showed in mice receiving 2 doses of Vi-TT (AlOH) while <br /> <br /> for the vaccine without adjuvant Vi-DT (PBS) having a booster effect greater than <br /> <br /> the others unadjuvanted vaccine. The groups of mice vaccinated by Vi-TT (AlOH) <br /> <br /> and Vi-DT (PBS) expressed IL4 at higher levels than the other groups, which <br /> <br /> correlated positively with the high Anti-Vi IgG in these animals. In conclusion, <br /> <br /> this study showed that selecting the carrier protein is critical to determine the <br /> <br /> high quality of TCV. Therefore, based on anti-Vi production and cytokine <br /> <br /> expression, DT is the best alternative for produce high-quality TCV. In addition, <br /> <br /> the use of adjuvant in TCV could increase the immunogenicity of vaccine and <br /> <br /> adjuvant AlOH could be used to improve the vaccine using TT as carrier protein. <br />