| I. Study on impact of C.sinensis on HBV infectionClonorchiasis, caused by Clonorchis sinensis(C.sinensis), is one of the parasitic zoonoses. It is estimated that around 35 million infected people in Asia, among which around 15 million infected people were in China. Previous epidemiological data showed that clonorchiasis is endemic in southeast of China, especially in Guangdong province. The people become infected C.Sinensis by the consumption of raw or poorly cooked fish that contains C.sinensis metacercarise. C.sinensis infection results in various complications in the liver and biliary systems, mainly cholelithiasis, cholangitis and cholecystitis. The adult worms of C.sinensis located in the smaller bile ducts of the liver that lead to cause mechanical damage; while excretory-secretory products(ESP) of C.sinensis lead to cause chemical damage. Both of mechanical damage and chemical damage together play a key role in causing hyperplasia and adenomatous changes in the bile duct. The ESP are known to involve in parasite, and clinical significance in the diagnosis and pathogenesis.Hepatitis B virus(HBV) infection is a major public health issue that may develop into cirrhosis, hepatic decompensation and hepatocellular carcinoma(HCC). An estimated 2 billion people have been infected, with more than 350 million are chronic carriers of the virus despite the availability of a prophylactic vaccine. HBV infection situation is even more severe in China and approximately 170 million people in China are chronically infected with HBV. Th1 responses seem to be involved in the clearance of HBV, while chronic HBV infection elicits very weak T cell responses.Even a relatively smaller population is infected with C.sinensis as compared with HBV, concurrent infection between HBV B virus and C.sinensis is often observed in some areas where C.sinensis is endemic. In some epidemiological studies, the positive rate of HBs Ag were significantly higher in endemic areas of C.sinensis than nonendemic areas. However, C.sinensisinfection and chronic HBV infection are two different causes of liver disease in China, few data currently exist as to whether there is any relation between HBV and C.sinensisinfection. There is no clear discussion whether the C.sinensis infections have impacts upon HBV infection or vice versa. ObjectivesThe aim of our study is to evaluate the impact of C.sinensisinfection on HBV infection as well as the response to antiviral therapy in co-infection patients and its related immunological mechanism. Methods and results 1. Criteria of cases selectionFirst we evaluated HBV patient by screening all consecutive patients in Third Affiliated hospital Sun Yat-Sen Univeristy from July 2014 to February 2015. The inclusion criteria were the following: men and women aged 18 years and older; HBV surface-antigen(HBs Ag)-positive; HBV DNA>20 IU/ml. Then co-infected patients were defined as having eggs in stool. Patients with the following concomitant conditions were excluded: those co-infected with HIV, hepatitis A,C,D and E, and those with alcoholic liver, autoimmune disease, cholestasis, serious heart diseases and pregnant women. 2. Comparison of liver function between co-infected and mono HBV infection patientsDuring the study period, there were 624 patients who met the selection criteria, of whom 51 were co-infection clonorchiasis, of whom 53 were mono C.sinensisinfected and HBV and 520 were HBV mono-infected. Those person who were HBs Ag negative and C.sinensis eggs negative were health control. Patients in three three groups were mostly men. Liver function was assessed by measurement of TB, ALT and AST in plasma. ALT, AST and TB levels were significantly higher in the co-infected group. Furthermore, HBV DNA log copies were also significantly higher in the co-infected patient group(p<0.05). Taken together, these data indicates that coinfection clonorchiasis and HBV had weaker liver function than mono-HBV infected and, that presence of C.sinensis might aggravate disease state. 3. Evaluate the relationship between HBV DNA and infection degreeThe C.sinensis infection degree are diagnose with numbers of eggs in stool. Coinfected patients have higher levels of HBV DNA, we further evaluate the relationship between HBV DNA and C.sinensis infection degree. Results showed that there was no apparent association between HBV DNA and numbers of C.sinensis eggs in stool when co-infected patients were C.sinensismild infection, however, those co-infected patients who have HBV DNA smaller than 106 have higher numbers of C.sinensis eggs were higher than those who have HBV DNA smaller than 106. 4. Evaluate the impact of C.sinensisinfection on the response to antiviral therapy in co-infection patientsGiven that presence of C.sinensis might aggravate HBV infection disease state, we further investigated whether removal of C.sinensis would have influence on efficacy of antiviral treatment in co-infected patient clinically. After one program of treatment, no significant differences were observed in levels of ALT and AST between two groups, however, C.sinesis /HBV-PZQ patients displayed significantly lower levels of TB than C.sinesis /HBV-NONPZQ patients(P<0.01). Additionally, C.sinesis /HBV-PZQ patients had lower levels of HBV DNA log copies compared to C.sinesis /HBV-NONPZQ(p<0.05). These results together suggested that combination of using antiviral drug with anti-clonorchiasis in co-infected patient would contribute to viral clearance and help with liver function recovery. 5. Evaluate the the impact of ESP on hepatitis B virus replicationCo-infected patients not only showed weaker liver function but also had significantly higher HBV DNA copies clinically. We reasoned that some metabolites of C.S may be directly enhance HBV replication. To address this possibility, we tested HBV DNA copies in supernatant after co-cultured with ESP and HBV positive patient serum in PBMC. HBV positive patient serum alone and ESP alone as a control, respectively. HBV DNA was measured by real-time PCR with lower detection limit of 20 IU/m L. As expected, HBV DNA copies were significantly higher in the culture supernatants from the PBMC co-cultured with ESP and HBV mixture than control groups(p<0.01). This data suggested that ESPs may, in some ways, promote viral replication. 6. Levels of cytokine secreted from stimulated PBMCIn order to define and compare Th1 cytokine(IL-2 and IFN-?) and Th2 cytokine(IL-4, IL-6 and IL-10) secretion following in vitro stimulation, we used quantitative RT-PCR and cytokine ELISA to analyze the levels of each cytokine secreted from PBMC, which were stimulated with ESP alone, HBV positive patient serum alone or the mixture of two, respectively. Data have been normalized for ?-actin transcript expression. Results showed that the levels of different cytokine m RNA varied evidently. IL-4 and IL-10 cytokine m RNA levels were higher in ESP-stimulated PBMC than in HBV-stimulated PBMC, whereas there is no significant difference in IL-2 and IFN-? cytokine m RNA levels between these two groups. In particularly, IL-6 cytokine m RNA levels were significantly higher in HBV positive sera stimulated PBMC than in ESPs stimulated PBMC. This suggests that both of stimulators could induce PBMC to produce Th1 and Th2 cytokines in vitro. Furthermore, in response to the treatment with mixture of HBV and ESP, IL-4 and IL-10 m RNA level increased two-fold and IL-6 m RNA level increased threefold comparing with PBMC stimulated with HBV positive sera alone, while there were no significant change in IL-2 and IFN-? level. Cytokine ELISA showed that IL-4, IFN-?, IL-10 and IL-13 level were higher in mixture of HBV and ESP-stimulated PBMC than in HBV-stimulated PBMC. These data suggested that PBMC stimulated by mixture of ESP and HBV mainly produced Th2 cytokines. 7. Cytokine expression level in sera from co-infected patientWe compared cytokine expression level in co-infected patients, mono-HBV infected patient and mono-C.sinensis infected patient, heatlh person as a control. Results showed that co-infected patient have a lower levels of IFN-? than mono-HBV infected patient and mono-C.s infected patient, but have a higher levels of IL-10 and IL-6 than mono-HBV infected patient. Theses indicated that co-infected patients expressed Th2 cytokine mainly. 8. ConclusionsOur results showed that co-infected individuals presented weaker liver function and higher HBV DNA titers. In co-infected patients, the efficacy of HBV treatment was associated with expelling worms. Possible reason for higher HBV DNA copies and weaker response to antiviral therapies in co-infected patients was that the shift in cytokine production from Th1 to Th2 that might inhibit viral clearance. II. Study on immune response and protective efficacy of DNA vaccine of EBOLA virusEbola virus(EBOV) is an enveloped single-stranded negative-sense RNA virus in the order Mononegavirales, which along with the Marburg virus(MARV) forms the Filovirus family. EBOV is the etiologic agent of Ebola Hemorrhagic Fever(EHF), a highly lethal hemorrhagic fever with up to 90% mortality.The genome of Ebola virus contains seven genes that encode 8 protein products. The 4th gene in Ebola virus genome contains 2 overlapping ORFs encoding s GP and GP. The wild type gene encode the s GP protein, which is transcribed at about 80% of the time. The GP is produced by a frameshift in the GP gene during m RNA transcription, which is transcribed at 20% of the time. The whole GP of Ebola virus is a transmembrane protein that forms trimers on cell surfaces and viral surfaces. It serves as viral membrane glycoprotein and mediates virus entry and infection of cells. The s GP is a secreted protein. It forms homodimers and is efficiently secreted from cells after synthesis. Although the s GP is synthesized and secreted in large quantities, its function in virus infection and pathogenesis is still unresolved. ObjectivesThe aim of our study is to study related mechanism of s GP in Ebola virus infection. Methods and results 1. Immunogenicity of EBOV GP editing site mutant DNA construct.We generated EBOV GP editing site mutants by using point mutation kits. GP-8A was made by inserting an 8th A into the wild type(GP-7A) editing site, resulting in GP1,2 as the dominant gene product. Silent ARG mutations were introduced into the GP-8A editing site to ablate transcriptional slippage, resulting in GP1,2 edit, that expresses GP1,2 as the sole gene product. The same mutations were also introduced into GP- 7A to generate s GPEdit that expresses s GP as the sole gene product. These constructs were subcloned into a mammalian expression vector(p CAGGS) and protein expression was examined in both He La cells. Western blots results showed that mutation of the editing site has a significant effect on GP expression. Then we used above constructs to immunized BALB/c mice to investigated the immunogenicity of editing site mutant consctucts. ELISA results showed that GP1,2 edit developed the highest titer of anti-GP1,2 antibodies compared to other constructs. Mice immunized with constructs expressing predominantly s GP(GP-7A and s GPEdit) developed much higher titers of anti-s GP antibodies than mice immunized with constructs expressing predominantly GP1,2(GP-8A or GP 1,2Edit). 2. s GP can compete for binding of anti-GP1,2 antibodies induced by s GP but not by GP1,2Given that animals immunized by GP1,2 or s GP develop antibodies that preferentially bind to different GP isoforms, we performed Western blot analysis to determine if there is a difference in the linear epitopes targeted by antibodies in GP1,2versus s GP-immunized mice. Results showed that antisera from GP1,2-immunized mice reacted strongly with GP1,2 but only weakly with s GP. Anti-sera from s GP immunized mice reacted strongly with s GP, but only weakly with GP1,2. Then competition ELISA showed that s GP was unable to compete for binding of anti-GP1,2 antibodies from GP1,2immunized mice, s GP was able to efficiently compete for anti-GP1,2 antibodies from s GP immunized mice. This suggests that most linear epitopes targeted by anti-GP1,2 antibodies from GP1,2-immunized mice are unshared with s GP. 3. Analysis of neutralizing activityWe investigated whether there was a difference in the ability of antisera from the immunization groups to neutralize EBOV GP 1,2-mediated virus infection. First, we generated pseudoviruses by using an Env-deficient HIV backbone pseudotyped with Zaire EBOV GP1,2. The results indicated that Antisera from both groups were able to effectivelyneutralize pseudoviruses, and antisera from GP1,2-immunized mice exhibited more potent neutralizing activity than antisera from s GP immunized mice. Then we performed competition ELISA to check whether s GP interferes with neutralization. s GP was able to completely attenuate neutralizing activity of antisera from s GPimmunized mice, while it had no effect on neutralizing activity of antisera from GP1,2-immunized mice. 4. Protective efficacy of different immunization regimens against lethal ZEBOV challenge.In order to check the efficacy of different immunization regimens, mice were immunization with 0 ?g of DNA vaccine dissolved in 100 ?L of PBS by intramuscular injection, followed by injection of the same formulation in 4 weeks later. Blood samples were collected from the retro-orbital sinus under anesthesia at 2 weeks after each immunization. After the final immunization, mice were challenged by intraperitoneal injection with 1000plaque-forming units(approximately 30 000 50% lethal doses) of mouse-adapted ZEBOV diluted in PBS. After challenge, mice were monitored daily for weight changes and signs of disease. First we checked antibody responses in all group, ELISA results showed that a third immunization with s GP or GP DNA vaccine augmented the levels of anti-GP antibodies in mice that had been primed by s GP or GP DNA vaccine. The highest levels of anti-GP antibody response were detected in group 5 mice, which received 3 immunizations with GP DNA vaccine. In contrast, while s GP DNA vaccine also induced high levels of anti-s GP antibodies, GP DNA vaccine only induced low levels of anti-s GP antibodies, even after 3 immunizations. Nonetheless, levels of anti-s GP antibodies were effectively boosted by s GP DNAvaccine in mice that had been primed by GPDNA vaccine(group 6), and boosting with GP DNA vaccine in mice that had been primed by s GP DNA vaccine also augmented the levels of anti-s GP antibodies to higher levels. Neutralizing assay indicated that similar levels of neutralizing activities were detected in sera from mice that had received 2 immunizationswith s GP or GP DNA vaccine. However, additional boosting by the same s GP or GP DNA vaccine only increased sera neutralizing activity slightly On the other hand, additional boosting by the different DNA vaccine did not further enhance serum neutralizing activities. Challenge data showed that Only 1 of 5 mice in group 1 that received 2 immunizations with s GP DNA survived the challenge, whereas 3 mice died on day 5 after challenge and 1 mouse died on day 8 after challenge. All mice in other vaccinated groups survived the challenge. Weight loss and illness were observed in other groups except groups mice immunized with 3 times of GP DNA. 5. ConclusionMutation of the editing site has a significant effect on GP expression. GP1,2 and s GP could develop anti-GP and anti-s GP antibodies. Antibodies induced by GP1,2 and s GP have neutralizing activity. The levels of anti-GP antibody response is important to protect mice from Ebola virus challenge. |
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