| Acquired immune deficiency syndrome (AIDS) which is caused by the humanimmunodeficiency virus (HIV) is the number one threat to human health, but aneffective HIV vaccine has not been developed successfully.In this study, Norovirus Pparticle was employed as the platform to exhibit these broad neutralizing antibodyepitopes of gp41membrane-proximal external region (MPER), to present antigensand elicit humoral immune response. Results show that vaccines induced guinea pigsto produce MPER specificity antibodies, but only with binding activity. Immuneresponse were not enhanced after boosting with HIV chimeric influenza vaccine.However, the application potential of norovirus P particle in vaccine research can notbe neglected. In the other hand, the traditional method of live attenuated influenzavaccine is not stable enough. In the production process, well determination ofeffective infection dosage affects the effectiveness of the vaccine. Thus, we try todevelop a more efficient titeration method. In this study, the qPCR technology hasbeen employed to titrate trivalent live attenuated influenza vaccine. This methodshortening the time needed for virus titration, overcome the disadvantages oftraditional method.1. Study on Chimeric Norovirus P particles to Elicit HIV-1BroadlyNeutralizing AntibodyThe world’s scientists study on HIV vaccine and hope that through differentstrategies can achieve the desire of develope vaccine successful. These methodsinclude using different antigens, preparating different forms of vaccine and choosingdifferent forms of immunization strategy. Eliciting efficient broadly neutralizingantibodies (BnAbs) is an important goal that has yet to be achieved for humanimmunodeficiency type1(HIV-1) vaccine development. Especially the MPERspecific neutralizing antibody2F5and4E10has the characteristics of most potentneutralizing ability and widest neutralization respectively. They are the important targets for a vaccine development. However, the present studies shown that only a fewof vaccines, can induced neutralizing antibodies in animals. However these results cannot reproducible in the clinical trials. Neutralizing antibody induction is still a verydifficult task, especially for MPER specific broadly neutralizing antibody.Norovirus (NoV) P particle has been proved to have the same immunogenicity asVLP vaccine, can induce strong humoral immunity and cellular immuneresponse.Thus, NoV P particle can be used as the subunit vaccine against NoV. Inaddition, NoV P particle has other advantages:(1) NoV P particle has72loop, able toaccommodate different sizes of exogenous fragments and does not affect theformation of the particle. These antigen epitope can be presented out due to theseloops existing in the particle surface, it increase the chance of antigen exposure;(2)NoV P particle preparated by the E. coli prokaryotic expression system, this methodhas advantages of simple, high output and stable structure. Therefore, NoV P particlecan be used for the preparation of NoV and other pathogens chimeric vaccine.In this study, NoV P particles were introduced as a new platform to display theMPER epitope of HIV-1as a vaccine with the aim of enhancing immune responses inguinea pigs. The results showed that HIV-1chimeric P particles were capable ofinducing MPER-specific antibody responses with a high titer of2F5or4E10likeantibody, but these antibodies only have a weak neutralizing activity. Immuneresponse were not enhanced after boosting with HIV chimeric influenza vaccine.Even though these findings are consistent with other previous studies, failed todevelop an effective HIV vaccine. However, the application potential of norovirus Pparticle in vaccine research can not be neglected. This design provides a new strategyfor HIV-1vaccines. Above all, to induced broad neutralizing antibody vaccineresearch and development for the strategy of HIV-1is still faced with enormouschallenges.2. Study on qPCR assay for titrating trivalent live attenuated influenzavaccinesAt present, the seasonal flu vaccine includes inactivated vaccine and liveattenuated vaccine.Live attenuated influenza vaccine (LAIV) can mimicthe process ofvirus infection. Due to present different antigen epitope with natural structure to theimmune system, thus has a better immunogenicity. LAIV consists of two strains ofinfluenza A viruses (H1N1and H3N2) and influenza B virus strains. LAIV should bepreparation according to the WHO recommended seasonal vaccine strains in each year.Therefore, it is urgent to supply flu vaccine on time.In the process of vaccine production, detection of effective infection dose is alaborious process. And the results are related to the quality control and theeffectiveness of the vaccination. Therefore, it’s necessary to perform virus titration bya method with accurate and efficient. The traditional methods such as50%Egginfectious dose assay (EID50) poccess many defects:(1) Requires a long time fortiteration, EID50assay need2-3days;(2) Time-consuming and laborious;(3) Highlycost, need specific neutralizing antibody blocking effect and further analysis by HAassay;(4) Large difference between each titration, deviation in±0.5log EID50range;(5) Different virus with different sensitivity to chicken embryos and the sources ofchicken embryos is not safe.They are likely to affect the final result.Therefore, theestablishment of a more effective virus titeration method is of great significance.Real-time quantitative PCR (qPCR) technology has been widely used due to ithas the advantages of rapid, sensitive, repeatable and reduced contamination.Especially in the field of mRNA expression level studies, or virus genome DNA copynumber detection, transgenic copy number, gene identification and pathogen detection.In recent years, qPCR have been used in virus vaccine titration, such as measlesvaccine virus, rotavirus vaccines and pox virus vaccine, with the advantages of fastand accurate.In this study, qPCR technology is employed in the detection of trivalent LAIVtitre. Research includes:(1) Design of multiple qPCR specific primers and probes,optimize the reaction conditions, and verify the validity and reproducibility of thereaction;(2) Determine the critical time for virus harvest, influenza A virus detectedin18h, influenza B virus for12h. But for type B detection, there was no significantdifference between12h and18h. As a result, qPCR can realize the titeration oftrivalent LAIVs within24h;(3) Select different batches of vaccine, preform qPCRand EID50analysis. Though the results are consistent, but qPCR method is moresensitive and stable. Therefore, this qPCR detection method established in this studycan be an alternative method for the EID50assay to titrate LAIVs with the advantagesof specific, sensitive, accurate and fast. |
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