| The late protein L1is the main antigen of Human papillomavirus (HPV) vaccine development. Currently, two kinds prophylactic vaccines have been licensed to prevent HPV infection:HPV16/18bivalent VLP vaccine (Cervarix) and HPV6/11/16/18tetravalent VLP vaccine (Gardasil), Gardasil derived from L1expression in yeast and Cervarix from expression in baculovirus infected insect cells. Both vaccines are recommended for girls and women aged9-26years preventing cervical cancer. However, these vaccines have some disadvantages such as:the application is limited due to poor complication and high costing in the developing country; both vaccines induce good systemic immunity but fail to elicit effective mucosal immunity; these vaccines provide protection against two kinds of high risk HPV types and offer limited cross-protection against other important carcinogenic HPV. Therefore, it’s significant to develop cheap, convenient and broad-spectrum new type HPV vaccine.Although the hepatitis B vaccine is very effective at preventiing hepatitis B virus infection, our country is the high incidence areas of hepatitis B. Currently, recombinant Hepatitis B Vaccine used alone is low immunogenic, which need adjuvant to enhance the role.but the cellular immunity induced by aluminum adjuvant-contaning hepatitis B vaccine is very weak, and it tends to cause low and non response state in10%people with yeast derived hepatitis B vaccination. Moreover, the vaccine requires mlultiprejuctin, and swelling, itching and redness at the injection. So, developing new type of vaccine is critical to control of disease.In addition, mucosal vaccines have the following advantages:(a) vaccine delivery is simple and painless, and avoids the reuse of needles and syringes, which are potential sources of infection;(b) mucosal vaccines increase the compliance to vaccination;(c) cellular immunity and sIgA antibody in the genital regions could be elicited;(d) repeating intranasal immunization could broaden the antigen protective spectrum. In2007, in order to greatly enhance the immunity effect of the vaccines, Jin-di-ke Biological Research Institute, Beijing, China developed a novel and safe JY adjuvant system which is composed of chitosan and IL-2. The aim of this study was to investigate the effects of JY adjuvant on mucosal immunity of human papillomavirus (HPV) types16and18L1protein virus-like particles (VLPs), and observe the cross reactions among HPV types58,11and6after repeating intranasal immunization. In addition, the study investigates the effects of different doses of IL-2and chitosan as adjuvant on immune responses induced by nasal-spray hepatitis B vaccine, in BALB/C mice.1. The study of mucosal immunity of HPV types16and18L1VLPBALB/c mice were immunized with HPV type16,18L1protein with or without JY adjuvant for three times by intramuscular, vaginal and intranasal routes, every three weeks0,3and6, respectively. Serum samples were collected at weeks2,5and8, respectively, and determined for IgG titer. Splenic lymphocytes and washes of respiratory tract were collected at the eighth week and determined for cellular and mucosal immunities. Half of the mice immunized by the intranasal route were further immunized for ten times, and then the serum samples were collected at weeks14,20,26,32and38, and determined for cross reactions among HPVI6,18L1protein and HPV58,11and6.1.1Humoral immunity induced by HPV types16and18L1VLPAfter three times of intranasal,vaginal and intramuscular immunization, respectively, HPV16and18L1VLP induced HPV-specific humoral immunity. The highest titer of serum IgG could be induced by intramuscular immunization of L1protein, the second was intranasal groups, vaginal groups were the lowest. however, after subsequent seven times of intranasal immunization, the serea IgG titers of the HPV types16and18L1protein combined immunization plus with JY adjuvant reach the level of the third times intramuscular immunization. In addition, following intramuscular, vaginal and intranasal immunization, the sera IgG levels of the L1protein plus adjuvant JY immunized mice were higher than in the L1protein alone, which indicated JY adjuvant enhanced the humoral immunity induced by HPV16,18L1proteins.1.2Mucosal immunity induced by HPV types16and18L1VLPAfter three times of intranasal, vaginal and intramuscular immunization, respectively, intramuscular immunization of HPV16and18L1protein failed to induce sIgA antibody, whereas, delivered through the intranasal and vaginal routes, the sIgA can be detected.1.2.1Mucosal immunity induced by intranasal immunization of L1VLPAfter intranasal immunization, compared with control group, the sIgA level increased significantly in experimental groups (P<0.01). The sIgA concentration in washes of respiratory tract of mice immunized with adjuvant-containing HPV16L1, HPV18L1, HPV16and18L1combined immunization (27.26,30.06and26.54μg/mL, respectively) significantly increased than the adjuvant-free HPV16LI, HPV18L1, HPV16and18L1combined immunization groups (22.11,23.47and23.85μg/mL, respectively)(P<0.05), indicating that JY adjuvant can enhanced mucosal immunity.1.2.2Mucosal immunity induced by vaginal immunization of L1VLPAfter vaginal immunization, compared with control group, the sIgA level increased significantly in experimental groups (P<0.01). The sIgA concentration in washes of respiratory tract of mice immunized with adjuvant-containing HPV16L1, HPV18LI, HPV16and18L1combined immunization (23.28,31.99and29.34μg/mL, respectively) significantly increased than the adjuvant-free HPV16L1, HPV18LI, HPV16and18L1combined immunization (25.07,18.64and19.65μg/mL, respectively)(P<0.05), indicating that JY adjuvant can enhanced mucosal immunity induced with HPV types16and18L1VLP by vaginal route.1.3Cellular immunity induced by HPV16and18L1VLP After three times of intranasal, vaginal and intramuscular immunization, respectively, vaginal immunization of HPV16and18L1protein failed to induce cellular antibody, in contrast, when delivered via the intranasal and intramuscular route, the cellular immune response was detectable.1.3.1Cellular immunity induced by intranasal immunization of L1VLPCompared with control group, the number of spot forming cells increased significantly in experimental groups (P<0.01). When HPV16L1protein was used to stimulate splenic lymphocytes, the number of spot forming cells significantly increased in mice immunized with adjuvant-containing HPV16L1, HPV16and18L1combined immunization (176.8±55.75and180.2±31.39, respectively) than in those with adjuvant-free L1protein (91±5.25and93.6±13.01, respectively, P<0.01). When HPV18L1protein was used to stimulate splenic lymphocytes, the number of spot forming cells significantly increased in mice immunized with adjuvant-containing HPV18L1, HPV16and18L1combined immunization (132.2.8±11.43and249.8±51.51, respectively) than the adjuvant-free L1protein (90±4.95and109.8.6±10.18, respectively, P<0.01). The data mentioned above suggested that JY adjuvant significantly enhanced the cellular immunity of HPV16and18L1VLP by intranasal immunization.1.3.2Cellular immunity induced by intramuscular immunization of L1VLPCompared with control group, the number of spot forming cells increased in experimental groups (P<0.05). When HPV16L1protein was used to stimulate splenic lymphocytes, the number of spot forming cells was slightly higher in mice immunized with adjuvant-containing HPV16L1, HPV16and18L1combined immunization (80.33±12.7and94.33±33.26, respectively) than the adjuvant-free L1protein group(75.67±10.41and74.33±13.61, respectively). When HPV18LI protein was used to stimulate splenic lymphocytes, the number of spot forming cells was slightly higher in mice immunized with adjuvant-containing HPV18L1and HPV16and18L1combined immunization (90.67±3.51and100±3.75, respectively) than the adjuvant-free L1protein (68±15.87and83.33±29.74, respectively)group, indicating that JY adjuvant enhanced the cellular immunity of HPV16and18LI VLP by intramuscular route.1.4Neutralizing antibody detection of HPV types16and18L1VLP Based on HPV16pseudovirus, after three times of intranasal and intravaginal immunization, mice immunized with HPV16and18L1protein combined immunization plus with JY adjuvant responded with titers of neutralizing antibody are1:100and1:50, respectively. However, after three times of intramuscular immunization, mice immunized with HPV types16and18L1protein combined immunization with or without JY adjuvant produced neutralizing antibody (1:6400,1:200, respectively), indicating that the titers of neutralizing antibody in mice intramuscular immunized with adjuvant-containing L1protein significantly increased than the adjuvant-free L1protein group. Our research shows that JY adjuvant could induce origanism to produce neutralizing antibody and improve the titer of antidody.1.5Immune cross reactivity of HPV types16and18L1VLP by repeating intranasal immunizationAnalysis showed that the sera of mice immunized with HPV types16and18L1protein showed cross-reaction with HPV58,11and6, and the serum specific IgG titer reached the higtest peak at the32nd week. In detail, the serum derived from intranasal immunization with HPV16L1protein plus adjuvant JY could react with HPV58, and the IgG titer reach1:131072at the32nd week, but1:8192and1:16384when react with HPV types6and11L1protein, respectively. Moreover, the serum derived from intranasal immunization with HPV18L1protein plus adjuvant JY could react with HPV58, and the IgG titer reach1:16384at weeks32, but both1:8192when react with HPV types6and11L1protein. These results showed HPV58had the highest cross reactivity with HPV16and18L1protein, but HPV11and6had somewhat lower cross reaction with HPV types16and18L1protein. In addition, the homology of HPV58L1compared with HPV16and18is76%and70%, respectively, but for HPV6and11L1are among66%-69%, therefore, for HPV16and18L1protein, the cross-reactivity with HPV58is superior to HPV11and6L1protein. Other research showed that antigen multiple immunizations can expand virus epitope recognition and lead to a response against non-dominant determinants, which provide immune protection against heterogenic virus, which is consistent with my conclusion.In conclusion, repeating intranasal immunization with HPV16and18L1protein broadened the antigen protective spectrum. Moreover, JY adjuvant enhanced the cellular immunity and sIgA level as well as humoral immunity induced with HPV types16and18L1protein by intranasal route, and also improved the humoral and mucosal immunity response induced by HPV16and18L1protein by vaginal route, and also improved the humoral and cellular immunity response induced by HPV16and18L1protein by intranasal route, indicating that JY adjuvant is a promising adjuvant.2. The study of mucosal immunity of Hepatitits B vaccineThe adjuvant composed by the different volume of IL-2and chitosan was used to hepatitis B virus surface antigen(HBsAg) vaccine, and then detect the IgG level in the mice serum immuned through intranasal routes and obtain the quantity of20000IU IL-2with0.025%chitosan used to hepatitis B virus surface antigen(HBsAg) vaccine have the highest IgG level. In addition, after hepatitis B vaccine containing aluminium adjuvant was immuned by injection, JY adjuvant hepatitis B vaccine for mucosal strengthen immune study indicated0.5ug or2μg containing aluminium hepatitis B vaccine intramuscular injection in primary or immune twice,8μg and4μg JY adjuvant hepatitis B vaccine strengthening immune can replace the second or third intramuscular immunization. The hepatitis B vaccine containing2μg aluminium intramuscular injection in primary or immune twice, the twice and third aluminium adjuvant intramuscular strengthening immunization group induced the same IgG level with the group of8μg and4μg JY adjuvant hepatitis B vaccine strengthen immunity. After0.5ug containing aluminium hepatitis B vaccine intramuscular injection in primary or immune twice, using0.5ug containing aluminium hepatitis B vaccine strengthening immune induced the same IgG level with the group of JY adjuvant hepatitis B vaccine strengthening immune. Thus JY adjuvant is safty and effective and can be used to develop the serum IgG level of the hepatitis B vaccine mucosal immune. |
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