STUDIES ON RIFT VALLEY FEVER VACCINE ADJUVANTED WITH ALUMINIUM PHOSPHATE | ||||
Assiut Veterinary Medical Journal | ||||
Article 14, Volume 52, Issue 110 - Serial Number 3, July 2006, Page 158-171 PDF (3.55 MB) | ||||
Document Type: Research article | ||||
DOI: 10.21608/avmj.2006.177392 | ||||
View on SCiNiTO | ||||
Author | ||||
LILY SOBHY SALAMA | ||||
Veterinary Serum and Vaccine Research Institute, Abbasia, Cairo | ||||
Abstract | ||||
Seventeen Balady sheep were used in this investigation. The animals were divided into 6 groups, the first group (G1) was vaccinated subcutaneously (S/C) with binary inactivated Rift Vally Fever (RVF) vaccine with 1.00% aluminium phosphate, the second group (G2) was vaccinated S/C with binary inactivated RVF vaccine with 0.75% aluminium phosphate, the third group (G3) was vaccinated S/C with binary inactivated RVF vaccine with 0.50% aluminium phosphate, the fourth group (G4) was vaccinated S/C with binary inactivated RVF vaccine with 0.25% aluminium phosphate, the fifth group (G5) was vaccinated S/C with inactivated RVF vaccine with 2% aluminium hydroxide gel while the sixth group (G6) left as control. The results revealed that group (1) followed by group (2) gave higher level of antibody and reaching its protective level earlier than RVF inactivated vaccine with aluminium gel and the duration of immunity of aluminium phosphate when compared with that of aluminium hydroxide is mush longer. In addition, the best vaccine was RVF inactivated vaccine containing 1.00% aluminium phosphate followed by RVF inactivated vaccine containing 0.75% aluminium phosphate as they gave higher level of antibody all over the period of the test compared with that of other vaccinated groups when tested by serum neutralization test and ELISA test. Moreover, manufacturing of these vaccines is easy to be done and of low cost. | ||||
Keywords | ||||
Key words: Rift valley fever; vaccination; virology | ||||
Full Text | ||||
Assut Vet. Med. J. Vol. 52 No. 110 July 2006 Veterinary Serum and Vaccine Research Institute, Abbasia, Cairo. STUDIES ON RIFT VALLEY FEVER VACCINE ADJUVANTED WITH ALUMINIUM PHOSPHATE (With 4 Tables and 2 Figures) By LILY SOBHY SALAMA (Received at 2/5/2006) دراسات على لقاح حمى الوادي المتصدع المثبط والمحسن باستخدام فوسفات الألومنيوم اللي صبحي سلامة استخدم في هذا البحث عدد سبعة عشر من الأغنام تم تقسيمها إلى ستة مجموعات. المجموعة الأولى تم تحصينها بلقاح حمى الوادى المتصدع المثبط بالبينارى ومضاف اليه فوسفات الالمونيوم والمجموعة الثانية تم تحصينها بلقاح حمى الوادى المتصدع المثبط %۱ اليه ۰٫۷۵% فوسفات الالومنيوم، والمجموعة الثالثة تم تحصينها بلقاح بالبينارى ومضاف % فوسفات الالومنيوم، ۰,۰۰ حمى الوادى المتصدع المثبط بالبينارى ومضاف اليه والمجموعة الرابعة تم تحصينها بلقاح حمى الوادى المتصدع المثبط بالبينارى ومضاف اليه %۰٫۲۰ فوسفات الالومنيوم. أما المجموعة الخامسة فقد تم تحصينها بلقاح حمى الوادي المتصدع المثبط بالبیناری ومضاف اليه الالومنيوم هیدروکسید جيل بينما المجموعة السادسة ترکت کضابط للتجربة. هذا وقد أظهرت النتائج أن اللقاح المضاف اليه 1%، ۰٫۷۵% فوسفات الألومنيوم أعطى أحسن النتائج وأعلى مستوى مناعی طوال فترة التجربة بالمقارنة بالمجموعات الأخرى عند استخدام تجربتي التعادل المصلي والإليزا. SUMMARY Seventeen Balady sheep were used in this investigation. The animals were divided into 6 groups, the first group (G1) was vaccinated subcutaneously (S/C) with binary inactivated Rift Vally Fever (RVF) vaccine with 1.00% aluminium phosphate, the second group (G2) was vaccinated S/C with binary inactivated RVF vaccine with 0.75% aluminium phosphate, the third group (G3) was vaccinated S/C with binary inactivated RVF vaccine with 0.50% aluminium phosphate, the fourth group (G4) was vaccinated S/C with binary inactivated RVF vaccine with 0.25% aluminium phosphate, the fifth group (G5) was vaccinated S/C with inactivated RVF vaccine with 2% aluminium 158 Assut Vet. Med. J. Vol. 52 No. 110 July 2006 hydroxide gel while the sixth group (G6) left as control. The results revealed that group (1) followed by group (2) gave higher level of antibody and reaching its protective level earlier than RVF inactivated vaccine with aluminium gel and the duration of immunity of aluminium phosphate when compared with that of aluminium hydroxide is mush longer. In addition, the best vaccine was RVF inactivated vaccine containing 1.00% aluminium phosphate followed by RVF inactivated vaccine containing 0.75% aluminium phosphate as they gave higher level of antibody all over the period of the test compared with that of other vaccinated groups when tested by serum neutralization test and ELISA test. Moreover, manufacturing of these vaccines is easy to be done and of low cost. Key words: Rift valley fever, vaccination, virology INTRODUCTION Rift valley fever virus (RVFV) is a Phlebovirus of the Bunyaviridae RNA single stranded virus family (WHO, 1982 and Connie, 1996). It is an acute or peracute mosquito-borne viral disease, most severe in sheep, cattle and goats, causing high mortalities in neonates and abortion in pregnant animals (Swanepoel and Goetzer, 1994). It emerges periodically throughout Africa, causing major threat for animal and human populations. The disease is widely distributed in Africa and Asia causes heavy losses among lambs and calves (Woods et al., 2002 and Fagbo, 2002). RVF was introduced to Egypt through importation of infected ruminants or camels from Sudan (Imam et al., 1977 and Sellers et al., 1982) and its reappearance in 1993 (El-Gabery et al., 1994) encouraged the authorities to develop a potent inactivated RVF vaccine. The prime purpose of vaccination is the induction of a high level of induced immunity among domestic animals which will serve to reduce the proportion of available hosts for amplification of the virus and may limit the extent of epizootics (Davies and Karstad, 1981). The Egyptian veterinary researchers succeeded in preparing a safe and potent alum adjuvant inactivated RVF vaccine to protect sheep and cattle against the disease (El-Nimr, 1980). Other studies were conducted by Taha et al. (1984) to improve the vaccine quality and to raise its efficiency. Aluminium adjuvants have been used for more than 50 years. There are three general types of aluminium containing adjuvants 159 Assut Vet. Med. J. Vol. 52 No. 110 July 2006 (aluminium hydroxide, aluminium phosphate and potassium aluminium sulphate (often called "alum"). The adjuvant effect of aluminium is manifested primarily by an increase in IgG and a delay in the rate of absorption of the precipitated antigen (Glenny et al., 1926). Aluminium adjuvanted antigen is rapidly encapsulated into a granuloma thus excluding it from the antibody producing mechanisms. It also increases trapping of lymphocytes in regional lymph nodes, thereby providing more cells for an enhanced immune response (Dresser et al., 1970, Taub et al., 1970). Aluminium compounds induce local granulomas which are rich in macrophages. Plasma cells are also present in the granuloma when an antigen is bound to the aluminium (White et al., 1955). It has been shown that aluminium will activate complement which may in turn activate macrophages and increase their phagocytic activity (Ramanthan et al., 1979). Also, aluminium salts attract esinophils to the site of injection and stimulate IgE antibody production (Kishimoto and Ishizaka, 1973). The aim of this work is to study the effect of aluminium phosphate as one of aluminium adjuvants when added to RVF binary inactivated virus on the immune response of vaccinated sheep. MATERIALS and METHODS Animals: 1. Mice (Swiss albino mice): 1.1. Adult mice: 21-28 days old mice were used for toxicity and potency tests for both aluminium phosphate and vaccines respectively. 1.2. Baby mice: 3-5 days old mice were used for safety test of the prepared inactivated virus. 2. Guinea pigs: Healthy adult guinea pigs of about 500 grams body weight were used for toxicity of aluminium phosphate. 3. Sheep: 3.1. Seventeen susceptible balady sheep about six months of age were used for evaluation of the immune response to the prepared vaccines, 3.2. Lambs: Twelve lambs of 5-10 days old were used for safety of the RVF vaccine with different aluminium phosphate concentrations together with the aluminium hydroxide gel (traditional one). 160 Assut Vet. Med. J. Vol. 52 No. 110 July 2006 Virus: RVF ZH-501 with a titre of 7.5 log10 TCID50/ml was kindly supplied by RVF Department, Veterinary Serum and Vaccine Research Institute, Abbasia, Cairo. Conjugate: Horseradish peroxidase conjugate labeled antisheep IgG was purchased from Sigma Company. It was used for ELISA test. Adjuvant: 1. Aluminium hydroxide gel: 2% aluminium hydroxide gel was purchased from Honil Limited, London, United Kingdom. 2. Aluminium phosphate: It is composed of: Di-natrium hydrogen phosphate-2 hydrte (Riedel de Haen) Natrium dihydrogen phosphate-Dihydrate (Merck) Aluminium potassium sulphate-12 hydrate technical grade (Ubichem) It was prepared according to Suhag Shirodkar et al. (1990). Toxicity test: A. Adult mice: They were used for the toxicity test of aluminium phosphate adjuvant in vaccine preparation. Each concentration was inoculated into two groups of mice (eight mice in each group) one group I/P and the second S/C and there was a group of eight mice was kept as a control and all groups were observed for 15 days post inoculation. B. Guinea pigs: Healthy adult guinea pigs each of about 500 grams body weight were used for the toxicity test of aluminium phosphate adjuvant in vaccine preparation. Each concentration was inoculated into two groups of guinea pigs (three guinea pigs in each group) one group I/P and the second S/C and a group of 3 guinea pigs was kept as a control and all groups were observed for 15 days post inoculation. Preparation of the vaccine: 1. Virus: RVF virus ZH-501 was inactivated by binary ethyleneimine according to Eman (1995). 2. Addition of aluminium phosphate: Alurninium phosphate was added with different concentrations to four portions of the inactivated virus as (1.00%, 0.75%, 0.50% and 0.25%) respectively. Healthy 161 Assut Vet. Med. J. Vol. 52 No. 110 July 2006 3. Addition of aluminium hydroxide: 2% aluminium hydroxide was added to the inactivated virus. 4. Evaluation of the vaccine: Sterility, safety and potency tests were performed on the different forms of vaccine according to protocol of OIE (2004). Experimental Design: Seventeen susceptible balady sheep were divided into 6 groups: Group 1: Three sheep were vaccinated S/C with Iml 10' TCID 50 inactivated RVF vaccine containing 1.00% aluminium phosphate. Group 2: Three sheep were vaccinated S/C with 1ml 107 TCID 50 inactivated RVF vaccine containing 0.75% aluminium phosphate. Group 3: Three sheep were vaccinated S/C with 1ml 107 TCID50 inactivated RVF vaccine containing 0.50% aluminium phosphate. Group 4: Three sheep were vaccinated S/C with Iml 107 TCID 50 inactivated RVF vaccine containing 0.25% aluminium phosphate. Group 5: Three sheep were vaccinated S/C with 1ml 107 TCID 50 inactivated RVF vaccine containing 2% aluminium hydroxide (traditional one). Group 6: Two sheep were kept as control (Not-vaccinated). All animals were observed for 6 months post vaccination for seroconversion. Serological tests: 1. Serum neutralization test: It was done according to Walker (1975). 2. Indirect enzyme linked immunosorbent assay (Indirect ELISA): It was done according to Voller et al. (1976). 162 Assut Vet. Med. J. Vol. 52 No. 110 July 2006 RESULTS Table 1: Results of toxicity test of aluminium phosphate in mice and guinea pigs Adjuvant Mice Guinea pigs Aluminium phosphate S/C I/P 1.00% 0/8 * 0/8 0/3 ** 0/3 S/C I/P 0.75 % 0/8 0/8 0/3 0/3 0.50 % 0/8 0/8 0/3 0/3 0.25% 0/8 0/8 0/3 0/3 Control 0/8 0/8 0/3 0/3 * Number of dead mice over number of survival mice. ** Number of dead guinea pigs over number of survived guinea pigs. Table 2: Results of sterility, safety and potency test of the prepared vaccines. Safety Forms of vaccine Sterility Baby mice Potency EDso/ml Lamb Sterile 0/6 0/2 0.0001/ml Sterile 0/6 0/2 0.0005/ml 0/6 0/2 0.0006/ml Aluminium phosphate 1.00% Aluminium phosphate 0.75% Aluminium phosphate 0.50% Aluminium phosphate 0.25% Aluminium hydroxide gel Control Sterile Sterile 0/6 0.0019/ml 0/2 0/2 Sterile 0/6 0.0008/ml 0/6 * 0/2 ** The minimum permissible limit of EDs/ml is 0.02/ml Safety test in baby mice = no signs of illness or death Safety test in lambs = no thermal or clinical reaction or manifestation * Control non-inoculated baby mice ** Control non-inoculated lambs 163 1.4 1.7 2.0 2.0 1.7 2.0 G1 2.4 3.0 0.9 1.9 2.7 2.4 1.5 2.5 NNNNN 0.4 164 Table 3: Result of neutralizing antibody index (NI) in sera of sheep vaccinated with different formula of RVF prepared vaccine Groups Neutralizing Indices Adjuvant No. of Before of Weeks post vaccination animals concentrations animals vaccination 4 8 1 12 20 0.4 0.7 2.4 | 2.7 2.4 2.0 Aluminium 0.3 1.0 2.4 2.7 3.0 2.4 phosphate 0.3 1.0 1.7 2.0 2.7 3.0 2.7 2.4 1.00% Mean 0.3 1.6 2.2 2.6 2.9 2.3 2.1 0.4 1.0 1.4 1.7 2.4 2.7 3.0 2.4 Aluminium 0.4 0.7 1.4 2.0 2.0 2.4 2.7 2.0 2.0 phosphate 0.7 1.0 1.7 1.7 2.0 2.4 2.4 2.4 2.0 0.75 % Mean 0.5 0.9 1.8 2.1 2.7 2.2 2.1 0.7 1.0 1.7 2.0 2.4 1.7 1.7 Aluminium 0.3 0.7 1.4 2.0 2.4 2.7 2.0 1.7 phosphate 0.4 1.0 1.4 2.0 2.4 2.4 2.4 2.0 0.50% Mean 0.8 1.2 2.2 2.6 2.0 1.8 0.3 1.0 1.7 2.0 2.4 1.7 1.4 Aluminium 0.3 1.0 1.4 1.7 2.0 2.0 1.7 1.7 phosphate 0.4 0.7 1.0 1.4 1.7 2.7 2.4 2.0 0.25% 1.7 Mean 0.3 0.8 1.1 1.5 2.1 1.8 1.6 0.3 0.7 1.0 1.4 1.7 2.0 2.4 2.0 1.7 Aluminium 0.3 0.7 1.4 2.0 2.4 2.7 2.4 2.0 hydroxide 0.4 1.4 1.7 2.0 2.7 2.7 (Alum gel) 2% 2.0 Mean 0.3 0.8 1.2 1.6 1.9 2.3 1.8 0.4 0.3 0.3 0.4 0.4 0.3 0.3 0.3 0.4 0.3 Control 0.3 0.4 0.3 0.3 0.4 0.4 0.4 0.3 0.3 0.3 Mcan 0.3 0.3 1 0.3 0.3 0.3 0.4 0.3 0.3 L 0.3 0.35 10.3 Assut Vet. Med. J. Vol. 52 No. 110 July 2006 0.3 1.9 0.7 2.0 G4 2.4 1.7 2.3 1.0 1.0 1.7 2.5 2.1 G6 * Protective level = 1.5 G1 phos Table 4: Result of indirect ELISA technique in sera of sheep vaccinated with different formula of RVF prepared vaccine Groups Optical Density Adjuvant No. of Before of Weeks post vaccination animals concentrations animals vaccination 8 12 16 20 24 0.035 0.071 0.076 0.083 0.091 10.101 0.116 0.093 0.089 0.080 Aluminium 0.038 0.067 0.079 0.085 0.094 0.100 0.120 0.096 0.090 0.086 phosphate 0.044 0.069 0.081 0.091 0.096 0.110 0.122 0.110 0.100 0.091 1.00 % Mean 0.039 0.069 0.078 0.086 0.093 0.103 0 .119 0.099 0.093 0.085 0.043 0.0690.073 10.081 0.087 0.093 0.101 0.091 0.083 0.079 Aluminium 3 0.040 0.066 0.069 10.079 1 0.084 0.098 10.112 0.098 0.085 0.077 phosphate 0.038 10.070 0.087 0.093 0.098 10.110 0.116 0.096 0.090 0.080 0.75 % Mean 0.040 0.068 0.076 0.084 0.089 0.100 0.109 0.095 0.086 0.078 0.038 0.066 0.071 0.079 0.083 10.096 0.106 0.098 0.085 0.069 Aluminium 0.035 0.069 0.071 0.0800.089 0.098 0.101 10.090 10.083 0.074 phosphate 0.040 0.067 0.079 0.084 0.086 0.091 0.098 0.086 0.080 0.078 0.50 % Mean 0.037 0.067 0.075 0.081 0.086 0.095 0.101 0.091 0.082 0.073 0.040 0.065 0.066 0.071 0.082 0.090 0.096 0.087 0.079 0.065 Aluninium 3 0.044 0.069 0.070 0.078 0.086 0.093 0.100 0.091 10.087 0.07-7 G4 phosphate 0.035 0.066 0.071 0.083 0.091 10.098 0.098 0.08210.073 0.069 0.25% 0.039 0.066 0.069 0.077 0.086 0.093 0.098 0.086 0.079 0.070 Aluminium 0.038 0.066 0.073 0.079 0.086 0.098 0.101 0.091 0.080 0.066 0.041 hydroxide 0.069 0.078 0.086 0.094 0.100 0.110 0.093 0.082 0.071 G5 (Alum gel) 0.044 0.071 0.080 10.084 0.091 0.100 | 0.106 0.094 0.087 0.077 2% Mean 0.041 0.077 0.083 0.090 0.099 0.105 0.092 0.083 0.071 0.040 0.033 0.042 0.040 0.035 0.038 0.042 0.035 0.040 0.038 Control 0.038 0.035 0.036 0.044 0.040 0.035 0.040 0.042 0.043 0.041 Mean 0.039 0.034 0.039 10.042 0.037 0.036 0.041 0.038 0.041 0.039 165 Assut Vet. Med. J. Vol. 52 No. 110 July 2006 Mean 0.068 G6 | * Cut-off value = 0.065 Assut Vet. Med. J. Vol. 52 No. 110 July 2006 Fig. (1): Neutralizing antibody index (NI) in sera of sheep vaccinated with different formula of RVF prepared vaccine Neutralizing Indice L Prevacc. 1 2 16 20 24 3 4 8 12 Weeks Post Vaccination - - Group (1) ------ Group (2) ------ Group (3) Group (4) * Group (5) - Control Fig. (2): Indirect ELISA technique in sera of sheep vaccinated with different formula of RVF prepared vaccine 0.14 0.12 0.1 Optical Density 0.08 0.06 0.04 0.02 - - - - Prevacc. 1 2 16 20 24 3 4 8 12 Weeks Post Vaccination - Group (1) on Group (4) 5. Group (2) - Group (3) * Group (5) ...... Control 166 Assut Vet. Med. J. Vol. 52 No. 110 July 2006 DISCUSSION Many studies were performed among adjuvants to improve RVF vaccine in order to increase its efficiency and duration of immune response. Aluminium containing adjuvants have historically served as immunopotentiators in vaccines and continue to be the most widely used adjuvants. Several aluminium compounds are used and are known as aluminium hydroxide adjuvant, aluminium phosphate adjuvant and potassium aluminium sulphate. Three potential mechanisms are frequently cited to explain how aluminium containing adjuvants increase antibody production. When the toxicity test was carried out in adult mice and guinea pigs the results revealed that neither S/C nor I/P routes of injection elicited any signs of toxicity during the test as shown in table 1. The different prepared formula of the prepared vaccines were sterile and safe when inoculated in baby mice and lambs which showed no signs of illness or deaths, also no elevation in body temperature in lambs. The most potent vaccine was that containing 1.00% aluminium phosphate as its ED50/ml was (0.0001/ml) followed by that containing 0.75% aluminium phosphate as its ED50/ml was (0.0005/ml) then followed by 0.50% aluminium phosphate as its ED50/ml was (0.0006/ml) and finally 0.25% aluminium phosphate as its ED50/ml was (0.0019/ml). All these batches were within the permissible limit as cited by Randall et (1964) who said that the ED50 must not more than 0.02/ml. The ED50/ml of aluminium hydroxide gel vaccine batch was 0.0008/ml). This could be explained by the fact that aluminium containing adjuvant and the adsorbed antigen remain at the site of injection. The antigen is released slowly to stimulate the production of antibodies (the depot mechanism), the aluminium containing adjuvants cause inflammation at the site of injection. Antigen presenting cells (APCs) are rapidly attracted to the site of inflammation because the antigen is also present at the site of injection, (APCs), encounter a high concentration of antigen (the inflammation mechanism). It has also been proposed that adsorption of antigen to aluminium containing adjuvants converts the soluble antigen to particular form. APCs can take up particular matter by phagocytosis. Thus, antigen which remains adsorbed, is taken into macrophages and dendritic cells. Thus aluminium containing adjuvants produce a high concentration of antigen within APCs, which results in immunopotentiation (Stanly et al., 2004). Aluminium phosphate is chemically amorphous in which some of the hydroxyl groups of 167 Assut Vet. Med. J. Vol. 52 No. 110 July 2006 aluminium hydroxide are replaced by phosphate groups (Shirodkar et al., 1990). Phosphate plays an important role as it keeps the adjuvant amorphous. The amorphous state is responsible for the high surface area and high adsorption capacity and being more soluble than aluminium hydroxide adjuvant as it is soluble in acid pH (< 4.0), in basic pH>8.0) and at neutral pH in solution of citric acid (Seeber et al., 1991 and Rinella et al., 1998). The immune response of vaccinated sheep was tested by SNT (Table 3). It was noticed that sera of sheep vaccinated with RVF vaccine with 1.00% aluminium phosphate (group 1) and 0.75% aluminium phosphate (group 2) reached the protective level at the 2nd week post vaccination being (1.6 NI, 1.5 NI, respectively) as Pini et al. (1973) suggested that the protective level was log 1.5 and reached its peak at the 12th week post vaccination with a mean of 2.9 NI, 2.7 NI, respectively). Animals of group 3 which was vaccinated with RVF vaccine containing 0.50% aluminium phosphate reached the protective level at the 3rd week post vaccination with an average of (1.6 NI), and reached its peak at the 12th week post vaccination with a mean of 2.6 NI, also animals of group (4) which vaccinated with RVF vaccine containing 0.25% aluminium phosphate reached the protective level at the 3rd week post vaccination with an average of (1.5 NI) and reached its peak at the 12" week post vaccination with a mean of 2.3 NI. From the previous data it was shown that (1.00% and 0.75% aluminium phosphate) are much better than the vaccine of aluminium hydroxide gel as it reached the protective level at the 3rd week post vaccination with an average of 1.6 NI and reached peak at the 12 week post vaccination with an average of 2.5 NI. The result of SNT was correlated with that obtained by ELISA as shown in table 4. This agreed with Eman (1995) and Hassan et al. (2001) but they used inactivated Rift valley fever vaccine adjuvanted with aluminium hydroxide gel as there is no available data on RVF vaccine adjuvanted with aluminium phosphate. From the previous data, aluminium phosphate induces immunological enhancement without toxicity and give high titre of antibody earlier than aluminium hydroxide gel especially when used in concentrations of 1.0% and 0.75%, and the duration of immunity of aluminium phosphate when compared with that of aluminium hydroxide is much longer. Also, it is easy to manufacture and of low cost. 168 Assut Vet. Med. J. Vol. 52 No. 110 July 2006 REFERENCES Connie, S.S. (1996): Bunyaviridae: The viruses and their replication. Field Virology. Third Edition, Vol. 1, Chapter 47. Philadelphia, Lippincott, Raven. Davies, F.G. and Karstad, L. (1981): Experimental infection of the African buffalo with the virus of Rift valley fever. Tropical Anim. Health and Production, 13 (4): 185-188. Dresser, D.W.; Taub, R.N. and Krantz, A.R. 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REFERENCES
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