| Dengue virus（DENV）belongs to the genus Flaviviridae of the family Flaviviridae,and is a non-segmented single-stranded positive-stranded RNA virus.Aedes aegypti and Aedes albopictus are the main vectors of its transmission.According to the different antigenicity of the main structural protein,Envelope protein（E）,it is divided into 4 serotypes,namely DENV-1,DENV-2,DENV-3 and DENV-4.Clinically,DENV mainly causes dengue fever（DF）,dengue hemorrhagic fever（DHF）and dengue shock syndrome（DSS）.Dengue fever caused by DENV has become a pandemic in many countries or regions in tropical and subtropical regions,and has become an important pathogenic microorganism that threatens local residents.About half of the world’s population lives in dengue-infected areas,and about 50 to 100 million population are infected with dengue virus every year,which has a great impact on the world economy and human health.At present,there are no specific drugs and vaccines for the treatment and prevention of dengue fever,and clinical,patients are mainly treated with symptomatic treatment.With the widespread clinical application of antibody drugs,the use of neutralizing antibodies to prevent and treat viruses has received more and more attention.However,antibody drugs cannot completely replace drugs and vaccines due to their short retention time in the body.Vectored Immunoprophylaxis（VIP）is a kind of passive immunization by vector-mediated delivery of genes encoding broadly neutralizing antibodies for in vivo expression.VIP is a new type of vaccine emerging in recent years,and has been shown to be effective in preventing infection with a variety of pathogens,especially those commonly found in travelers（including influenza A virus,Plasmodium falciparum,hepatitis C virus,respiratory Syncytial virus,Bacillus anthracis,Dengue virus and Chikungunya virus,etc.）.Unlike traditional vaccines that use antigens to trigger a host immune system response.Unlike traditional vaccines that use antigens to trigger a host immune system response,VIP vaccines bypass the humoral immune system and produce customized neutralizing antibodies directly from non-hematopoietic cells.The biggest advantage of the VIP vaccine is that it can express neutralizing antibodies stably and efficiently in the body for a long time,which solves the shortcomings of antibody drugs to the greatest extent.This study firstly conducted in-depth research and analysis on the causes,epidemic trends and characteristics of the dengue outbreak in Yunnan Province from 2017 to 2018;then,phage display technology was used to screen dengue broad-spectrum specific antibodies,and the screened antibodies were screened.Humanization optimization and biological activity identification were carried out;finally,recombinant VIP vaccine candidates based on adeno-associated virus（AAV）expression of different neutralizing antibodies were constructed,and immune research experiments were carried out.The main results are as follows:1)The epidemiological survey of DENV in Yunnan Province from 2017 to 2018 showed that the positive rates of DENV in 2017 and 2018 were 18.1%and 25.0%,respectively.The proportion of DENV-2 strains increased from 30.8%in 2017 to 52.0%in 2018.The geographic distribution and trend of increase and decrease of local cases and imported cases were significantly positively correlated,and the local strains had high homology and closest relatives with the strains from travelers of Laos and Myanmar.It was speculated that the dengue epidemic in Yunnan was mainly caused by imported cases.Dengue fever outbreaks are mainly caused by imported cases.A total of three complete genome sequences and seven E gene sequences were obtained by sequencing.DENV-1 isolates belong to genotype I and genotype V,and DENV-2isolates belong to Asian type I,Asian type II and Cosmopolitan type.Among them,the genotype I of DENV-1 and the Cosmopolitan type of DENV-2 were the main DENV strains prevalent in Yunnan,and the recombination analysis found that four strains had recombination phenomenon.2)A SYBR Green I real-time PCR detection method for 4 serotypes of DENV was established.The detection method has a good linear relationship in the range of 4.88×101～4.88×109 copies/μL,and the lowest detection limit is 4.88 copies/μL.It has the characteristics of excellent specificity and high repeatability.One DENV-1 strain（YN/RL,genotype I）and two DENV-2 strains（YN/MH and YN/017,Asian type II）were isolated,and the highest titers of the three strains were all greater than 107 copies/m L.Sequence analysis revealed that YN/017isolates lacked 13 nucleotides（10270 nt-10282 nt）in the 3’UTRs,resulting in significant changes in RNA secondary structure.3)Using the baculovirus expression system,the EDIII region of DENV1～4 E protein was successfully expressed,and high titer levels of specific antibodies were produced after immunizing mice.Using phage display technology,screening with high specificity for sequencing,and finally obtained 10 different sc Fv antibody sequences.The sc Fv antibody2H8B,which has strong affinity for the four antigens,was screened and verified by binding experiments with EDIII proteins of four serotypes.4)1A1D,2A10,2H8B and 1G6 antibody sequences were optimized for humanization.Four kinds of antibodies were expressed in vitro using CHO cells,and finally four kinds of purified humanized antibodies were obtained.The four antibodies have high affinity to E protein of different serotypes.The neutralization test results in vitro and in vivo showed that the 1A1D antibody had the best neutralizing activity against DENV-1 and DENV-3,with IC50 of 0.11×10-7M and 0.15×10-7M,respectively;2A10 antibody of 0.14×10-7M could neutralize 50%of the DENV-2;the strongest neutralizing ability to DENV-4 is 1G6,with IC50 of 0.09×10-7M.5)Based on four kinds of neutralizing antibodies optimized by humanization and AAV as the delivery platform,four kinds of VIP vaccine candidates including AAV-1A1D,AAV-1G6,AAV-2A10 and AAV-2H8B were constructed with AAV as the delivery platform.BALB/c mice were immunized with three doses of high,medium and low doses of the four VIP vaccine candidates.All experimental groups could produce high levels of antibodies,and the antibody levels were greater than 200μg/m L after 24 weeks.After mixed immunization of the four VIP vaccine candidates in different combinations,the antibody level in BALB/c mice was greater than 500μg/m L,and the antibody level could still be maintained at about 400μg/m L after 18-24 weeks.The results of neutralization test and challenge protection test showed that after 2 weeks of combined immunization,the neutralizing antibody titers of group N（1A1D+1G6+2A10）and group W（1A1D+1G6+2A10+2H8B）were both greater than 100,and the protective effect was greater than 60%;after 18-24 weeks of immunization,the neutralizing antibody titers of the N group against four different serotypes of DENV were all>320,and the protective effect was close to 90%.In summary,the DENV strains in Yunnan Province are constantly undergoing new changes,and the constant mutation of DENV and foreign input have brought great challenges to the prevention and control of dengue fever.This study enriched the current DENV molecular epidemiology data in Yunnan Province,a high-affinity broad-spectrum DENV antibody-2H8B was screened by phage screening technology,four VIP candidate vaccines were constructed,and neutralizing antibodies and challenge protection tests were all tested.Co-immunization was demonstrated to provide protection to mice.This study provides a new research idea for the study of dengue vaccine candidates. |
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