Study Of Hexon-chimaeric RAd5Vectors Evading Pre-existing Anti-vector Immunity

Many features make adenoviruses (Ads) attractive as vectors for gene delivery andcandidate vaccines. Recombinant, replication-incompetent adenovirus (rAd) vectorshave some desirable features for vector-based vaccines. Vector appears safe and doesnot persist in vivo; Scale-up feasibility and manufacturing capacity; Grows to high titersin suspension cell cultures; Does not integrate into the host cell genome; Efficienttransduction of mammalian cells; Highly immunogenic in animals and humans;Prime–boost strategies can augment responses. Because of these advantages, adenoviruswas extremely widely used in gene transduction, vaccination, gene therapy and someother fields.Currently, the most widely used adenovirus vectors is the human adenovirus type5,it is also the main viral vectors for cancer gene therapy and human immunodeficiencyvirus (HIV) vaccine clinical research. In addition, vaccines for hepatitis B virus, malaria,tuberculosis and other pathogens are also attached great importance to the application ofthe adenovirus type5vectors. However, a major problem of the recombinant adenovirustype5(rAd5) is the pre-existing anti-vector immunity. The high level of Ad5neutralizing antibodies (NAbs) titer will significantly reduce the transduction efficiencyand transgenic expression of the rAd5vectors. Epidemiology studies of humanadenovirus have shown that: approximately60-70%of the United States and Europepopulation has Ad5NAbs, whereas higher titers and frequencies as high as98%arefound in parts of the Sahara of Africa and Southeast Asia. Although domesticadenovirus vector used in cancer gene therapy and other related research, however, stilllacking epidemiological survey data of Ad5and some other rare serotypes. In additionto the pre-existing immunity, rAd5can be said that is an almost perfect vector of genetherapy and vaccine. Therefore, study on the mechanism of Ad5pre-existing immunityand develop rAd5vectors which will circumvent pre-existing anti-vector immunity hasa strong scientific significance and applied value. This study by analyzing the antigenic sites of Ad5neutralizing antibodies, and thenby genetically engineering for rAd5, that is using antigenic genes from rare adenovirusserotype to switch the rAd5and to construct the chimeric vectors, hoping to obtain annovel rAd5vector which is easy packaging, having strong immunogenicity and caneffectively prevent the preexisting anti-Ad5immunity.This paper first detected the Ad5neutralizing antibody titers using the standardizedneutralization assays by collecting more than1000human sera in China. The resultsshowed that72%of the health adults were seropositive to Ad5neutralizing antibody(titer>18) and close to Europe and the United States. In addition, the results alsoshowed that the level of Ad5seroprevalence in southern China was slightly higher thanother regions. To further analyze the situation of children become infected withadenovirus, we also detected the children serum samples from the northeast China.Results showed that children in7-12months had the lowest seroprevalence of Ad2andAd5NAbs, which are suitable candidates for adenovirus-based vaccines. Conducive tothe infections of some rare adenovirus serotypes in China, we established a fast,convenient, and accurate typing serological method to detect the adenovirus neutralizingantibodies in human serum by analysis the immunological properties of the adenovirusin human serum and prokaryotic expression of the adenovirus capsid protein functionaldomain. We showed that the seroprevalence of Ad37and Ad43in China is relativelylow, detected by our method for the sera from children in Northeast China.Subsequently, we analyzed the immunological properties of adenovirus on theprotein levels. We first used the Ad5HVRs and knob protein to analyze the adenoviruscapsid specific humoral immune response. We confirmed that the high-infected peopleand animals immunized with Ad5would mainly generate antibodies to hexon. Take intoaccount the difficulty of vector construction, HVRs chimeric protein were used toevaluate the neutralizing activity of hexon epitopes in protein level. Results showed thatHVR5and HVR7had only a part of hexon activity to neutralizing antibodies (NAbs)compared with the complete activity of HVR1–7. These results provided a basis for thetransformation of the following virus chimeric gene and strategy selection.To obtain easy packing vectors, we selectively constructed a total of14Loop1-2,HVRs and Loop4chimeric adenovirus backbone plasmids from three kinds of rareserotypes (Ad26, Ad37, and Ad43). Then the chimeric plasmids and the shuttle plasmidexpressed eGFP were co-transfected into HEK293cells under the same conditions to examine the packaging of the chimeric vectors. The results showed that Loop1-2andfull-length HVRs replacement plasmids were difficult in packaging, and the Loop4replacement plasmids had not improved the packaging capacity of the chimericplasmids. However, only HVR5and HVR7replacement (from Ad26, Ad37and Ad43)chimeric plasmids and as a positive control unmodified rAd5skeleton plasmidsuccessed to rescue viruses and showed a strong replication ability. We also found thatpackaging efficiency of the Ad43HVR5,7chimeric plasmid was significantly increased.Activity studies in vitro illustrated that the successful packaging chimeric vectors gotvery strong transgene expression and remained stable for at least15serial passages invitro without detectable loss of the transgene or changes to the chimaeric hexonsequence.Then we found that the immunogenicity of chimeric adenovirus vector for Ad5virus and the Gag gene were similar to rAd5-Gag by mice immunization. Finally, bysimulating on adenovirus NAbs level of human body in mouse model, we showed thatHVR5,7chimeric vectors in mice could escape a certain degree of pre-existingimmunity but difficult to avoid high levels of pre-existing immunity effects. We alsoshowed that certain levels of pre-existing immunity from other Ad serotypes did notaffect the immunogenicity of the rAd5vectors.In summary, this study showed that due to the complex nature of the immune ofadenovirus, chimeric transformation by the existing information may be difficult toobtain a vector to evade high levels of pre-existing immunity in humans. And thecomplex genetic engineering modification would cause the vector difficult to packaging.Therefore, it prompts us that further and in-depth study on the immune mechanism ofAd5would be possible to construct a more ideal gene therapy and vaccine vector.However, this study still have certain significance on adenovirus infection andadenoviral vector packaging and immunogenicity, and also provide some basis for thefurther application of the adenovirus vector in China.