Detection Of Adenovirus Type 5 Neutralizing Antibody In Common Marmosets

Adenoviruses are double-stranded DNA viruses isolated and cultured from adenoid tissue and reproduce mainly in nucleus. It is not only the common pathogen to infect respiratory, conjunctiva and intestinal systems, but also causes aseptic meningitis, encephalitis, myocarditis and hepatitis. Adenovirus has strong infectivity especially in children, the elderly and immunocompromised individuals like human immunodeficiency virus (HIV) infected and transplant patients. With the co-penetration and development of immunology, virology and molecular biology, adenovirus was widely explored in adenovirus infection, anti-adenovirus therapy and vectors for gene delivery and vaccine candidates.Viral vector vaccine is a kind of recombinant virus capable of expressing protective antigen gene by inserting the gene into the virus genome. This vaccine is made of live viruses, have native immunogenicity and no need for purification. It can produce specific protective immune response after in-vivo delivery, with the virus itself plays an adjuvant effect to enhance immunity. Adenovirus, adeno-associated virus, herpes simplex virus, pox virus, retrovirus, small RNA virus and baculovirus have been used in vaccine research, many of which have entered clinical human trials. Compared with other vectors, human adenoviral vectors have some desirable features for vector-based vaccines. Vector has scale-up feasibility and manufacturing capacity for carrying foreign genes; It’s Safe and stable; Mature production can be made with high titers in suspension culture; It’s capable to infect a variety of cell types including quiescent and splinter cell; It does not integrate into the host cell genome; It can induce mucosal and system immune response; It’s highly immunogenic in animals and humans; It has plentiful serotypes with high transduction efficiency. Because of these advantages, adenovirus makes extremely progression in vector-based gene therapy and vaccines.Currently, the most widely used adenoviral vectors is Human Mastadenovirus 5(AdHu5). AdHu5 is more immunogenic than other serotypes, so that was widely used as viral vectors for HIV, hepatitis B virus, malaria, tuberculosis and other pathogens. However, two decades of intensive research have also highlighted certain challenges associated with the use of AdHu5 vectors that limit their clinical applications. These include both a high innate immune toxicity profile associated with a marked liver tropism when AdHu5 vectors are delivered intravenously, and a worldwide high preexisting adaptive immunity against AdHu5 in man. The failure of rAd5/HIV vaccine in clinical trial indicated reduction of transfer efficacy and antigen gene expression in individuals with high pre-existing neutralizing antibodies to Ad5. However, AdHu5 vector vaccine can be evaluate for the immunogenicity and tolerance of the immunogen that cause the protective immunity in non-human primate models which has a homology of up to 99.9% with human beings. Thus, it is needed to detect the level of AdHu5 neutralizing antibody in the non-human primate that was proposed to be the model before clinical trial.In recent years, common marmoset (Callithrix jacchus), a kind of small new world primate has attracted considerable attentions as widely used in basic biology, pharmacology and toxicology research. Common marmoset is a new world primate belonging to the Callitrichidae family, native to the Atlantic coastal forests in northeastern Brazil. The lifespan of marmosets is relatively compact compared with other NHPs, while reaching maturity by 18-24 months of age, producing offspring by three years and reaching old stage by 8 years. The compressed lifespan of the marmoset is attractive in scientific research because the number of marmosets available for research can be scaled up quickly when the need arises and then naturally reduced when excessive numbers of animals are not needed. Adult common marmoset has a weight of 350-400g and gentle mind, which is suitable for breeding and well meets the experimental requirements. The marmoset has been shown to be an outstanding model for studying aging, reproduction, neuroscience, toxicology, and infectious disease. Undoubtedly, increased use of marmoset models will accelerate pre-clinical development of vaccines and therapeutics to highly pathogenic viruses. Research found that 31.3% of common marmoset residing in Brazil had antibodies that neutralized AdHu5 while such antibodies were not detected in marmoset residing in the USA, which may be related with environment they lived and creature they contact. However, it hadn’t been reported about the NAb level of common marmosets husbandry and breeding in China before. Our previous work was major in constructing HCV/GBVB chimeric virus infected marmoset model, which will be suitable to evaluate the adenoviral vector based HCV vaccine before clinical trial. Thus, it is significantly important to evaluate the immunogenicity and tolerance of target gene with Ad5 vector in the model, and firstly we should pre-test neutralizing antibody to AdHu5 in common marmosets.Objective:The study is to examine seroprevalence of adenovirus type 5 neutralizing antibody in common marmosets, and make a comparison of two different neutralizing tests of chemiluminescence and flow cytometry (FCM). Finally to evaluate the feasibility and applicability of using the common marmoset as a model of pre-clinical research of recombinant adenovirus type 5 vector vaccine, provide important background information for research and development of HCV vaccine product using in preclinical trials, and establish foundations for industrialization of HCV vaccine in our country.Method:Currently, two different assays are used to determine anti-Ad5 neutralizing activity in serum from human and animal. One of them is the readout performed microscopically by scoring the Ad-mediated cytopathic effect (CPE) after WT-Ad was neutralized by antibody. In another assay, the inhibition of transgene expression in replication-deficient Ad is taken as a parameter for antiviral neutralization. For such Ad transgene expression inhibition assays, rAds carrying luciferase, Zs Green (green fluorescent protein), or LacZ as reporter genes can be used. The results of CPE are highly dependent on the operator and the timing of readout, making it unsuitable for large scale serum within short time. In conclusion, we choose to test anti-Ad5 neutralizing activity through constructing recombinant replication-incompetent adenovirus type 5 (rAd5) carrying luciferase and Zs Green as reporter genes.We obtained the luciferase and Zs Green reporter gene by copying the plasmid pHAGE-CMV-Luc-IZs Green, and insert EcoR I and Nhe I restriction enzyme site in 51 and 3’ends of Luc-IZs Green by the means of primer design. The target sequence was insert into the expression vector pDC315 to construct the recombinant adenovirus shutter plasmid pDC315-Luc-IZs Green. By liposome transfection, we co-transduced the 293 A cell line which can complement E1 protein with a certain amount of packed adenovirus shuttle vectors and packing bone plasmid pBHGlox(delta)E1,3Cre. Through the repackage of 293A cells, infectious recombinant adenovirus rAd5/LUC/Zs Green which has integrated with luciferase and Zs Green genes was obtained. Monoclonal recombinant adenovirus was screened by 3 times plaque formation experiment. Large scale amplification of monoclonal virus was performed in 293A cell. After amplification, the virus was purified by cesium chloride density gradient centrifugation and the infection titer was tested according to 50% tissue culture infection dose (TCID50). In order to identify rAd5/LUC/Zs Green after purification, PCR or RT-PCR was used to amplify Luc-IZs Green from genomic DNA and total RNA. At the same time, the expression of the protein was verified by cell immunohistochemical experiment. Consequently, the optimal adenovirus virus infection titer was determined by detecting the expression efficiency of luciferase and Zs Green report gene when doing the neutralization experiment. Finally, a serial doubling dilution of serum from 25 common marmosets were made and then added to a number of VPs that was determined by the virus titration. The readout of green fluorescence and luciferase was correspondingly measured with FCM and chemiluminescence, which will compared with virus control to judge anti-Ad5 neutralizing antibody titer in marmosets.Results:1. Construction of shuttle plasmid. Successfully constructed the adenovirus shuttle vector pDC315-Luc-IZs Green containing luciferase and Zs Green reporter gene with conventional molecular cloning method, and preliminarily identified that these two plasmids can correctly express labeled proteins in eukaryotic cells 293A by using the method of transient transfection.2. Package of recombinant adenovirus and titer determination. By co-transfection of adenovirus shuttle vectors and packing bone plasmid to 293A, infectious recombinant adenovirus rAd5/LUC/Zs Green was successfully achieved. The infection titer finally reached 6.9 × 1011.5PFU/ml after 3 time plaque formation, large scale amplification and purification.3. Identification of recombinant adenovirus. Successfully amplified Luc-IZs Green gene from genomic DNA and total RNA by PCR or RT-PCR. Virus structure protein could express in 293A cell detecting by cell immunohistochemical experiment. 293 A was infected with a serial dilution of rAd5/LUC/Zs Green to get the expression efficiency of luciferase and Zs Green report gene. Results showed that the optimal adenovirus virus infection titer using in neutralization assay was between 6.9 X 106.5PFU/ml～6.9 × 107.5PFU/ml.4. Determine anti-Ad5 neutralizing activity in common marmosets. Neutralization determined by calculating the Zs Green expression inhibition through FCM showed 5 marmosets were positive,4 marmosets’ titer of NAb was 1/16 and only one was 1/64. For the other method, neutralization determined by luciferase expression inhibition through chemiluminescence showed 7 marmosets of 25 (28%) were anti-Ad5 neutralizing antibody (NAb) positive.4 of the Ad5 positive marmosets demonstrated low titers of 1/16. Two of them were 1/32 and another one was 1/128.5. Comparison of two different neutralizing assays. Statistical analysis showed the results of chemiluminescence method and flow cytometry test are consistent (P<0.01) overall with slightly difference. Through detailed comparison we think chemiluminescence method is more simple, sensitive, accurate and robust.Conclusions:(1) Recombinant adenovirus rAd5/LUC/Zs Green carrying luciferase and Zs Green report gene was packaged using ADMax adenovirus packaging system.(2) Anti-Ad5 neutralizing activity in 25 common marmosets from China was tested and it revealed that Ad5 seroprevalence of common marmosets was 20%-30%. Most of Ad5 positive marmosets demonstrated low titers<32, rare marmoset has higher NAb titers.(3) From the view of reproducibility, accuracy, robustness, simplicity, and sensitivity of the assay, we proposed a neutralization assay based on rAd5 carrying luciferase with readout in terms of the inhibition of luciferase transgene expression.(4) The results provided basic data for common marmosets which will play an important role of model for adenovirus type 5 or other rare serotype adenovirus based vaccination.