Seroprevalence Of Neutralizing Antibodies To Human Adenovirus In Healthy People And Identification Of Neutralizing Epitopes Of Human Adenovirus Type3by Monoclonal Antibody

BackgroundAdenovirus (Adv) infections spread through the whole world. It is strong infective and can cause respiratory infection, acute pharyngitis, epidemic conjunctivitis, gastrointestinal infection and other multi-system organ infections. HIV people and transplantation patient is likely to be infected by Adenovirus due to immunocompromised. Therefore, more and more researchers focus on adenovirus prevention, diagnosis and treatment.Human adenovirus (HAdVs) belongs to family Adenoviridae, genus Mastadenovirus. The57serotypes of HAdV have been places in seven species (A to G) by International Taxonomy of Viruses Commission (ICTV), depending on the immunological, biological and biochemical characteristics. In2011, the serotype58of adenovirus had been reported which have been submitted to the ICTV. Different serotypes of adenovirus could infect different tissue, which caused different clinical symptoms. HAdV-3and-7of Group B, and HAdV-4of Group E often cause symptoms such as fever, cough, pneumonia, pharyngeal conjunctival fever and other lower respiratory tract infections. In2004-2005, an outbreak and epidemic of HAdV- 3occurred in Jiangsu, Hubei, Jiangsu Province and Beijing; An outbreak of pharyngeal-conjunctival fever induced by HAdV-3occurred in Guangzhou in2004; An outbreak of severe lower respiratory tract symptoms with HAdV-7occurred in Shanxi Province in2009; In2008-2009, an outbreak of HAdV-3occurred in New Brunswick, Canada, with hundreds of people infected. The infections of HAdV-4spread in military trainees and a long-term care facility for elderly in the U.S.Similar other viral infections, Adenovirus infection cured by the resistance of own immune system, not by the effects of drugs. Previous studies have shown serum antibodies against adenovirus could prevent infection by the same serotype adenovirus. Therefore, the development of efficient, safe and low-cost adenovirus vaccine is the most effective way to prevent adenovirus infection. The introduction in1971of oral vaccines against adenovirus types4and7for use among military trainees in the U.S., dramatically reduced the incidence of adenovirus disease and febrile respiratory illnesses in that population. In1999, the vaccine stopped the supply. In subsequent years, the incidence of febrile respiratory illness increased markedly at basic training centers. In2011, the FDA approved the new oral Adenovirus Type4and Type7Vaccine to market. Subsequently, rates of febrile respiratory illnesses and adenovirus isolations markedly declined. But, the live oral vaccines were limited used in the military for the risks of infection and recombination. Therefore, development of safe and effective adenovirus vaccine need a long time.On the other hand, Adenoviruses are currently the most widely used vectors in gene therapy fields. In comparison with other viral vectors, adenovirus vectors have many virtues such as the high capacity to genes, not integrating into the genome of target cells, high-titer and stability, reproduction not only in the intestine but also in the respiratory tract, and with the prospects for the development of oral vaccine. Latest data show Adv2and Adv5vectors are a quarter of all vaccine vectors. Adenovirus vectors were limited to application by intensive host rejection. Recently, the research reports of Adv2and Adv5vectors show the two vectors would not to be used in cancer treatment for the adenovirus receptor CAR not expressed in the surface of many tumor cells. While, Adv3receptor could express in the surface of tumor cells. So the Adv3will likely be a new vector used in cancer treatment.Some researches of engineered vaccine against HAdV-3and-7have been reported by our research group. A replication-competent human Adv3-based vector was successfully constructed; a recombinant virus of Adv3packaged by Adv7hexon was constructed in2011. Recently, an Enterovirus71vaccine was constructed with neutralizing epitope incorporation within Adv3hexon. To the researches of adenovirus vaccine and adenovirus vector, it will be helpful to analysis prevalence of serum neutralizing antibodies against the Adv3and Adv7in the healthy people. Therefore, it is the part I of my work of the survey to the prevalence of serum neutralizing antibodies of Adenovirus in Guangzhou. The second part of my work is the study about preparation and identification monoclonal antibodies against Adv3. The third part of my work is analysis of neutralizing epitope of Adv3.Part I Seroprevalence of Neutralizing Antibodies to Human Adenovirus in Healthy PeopleA total of452sera from humans were collected and tested for serum NAb to HAdV-3,4and7. Serum samples were collected in2007, from Guangzhou Children’s Hospital and Guangzhou Bank hospital. Sera were obtained from adults who had a medical examination in the Guangzhou Bank Hospital, and children not exhibiting signs of respiratory tract infection in the Guangzhou Children’s Hospital. According age of people, the sera were grouped into seven sets:0-6month,7-12month,1-3year,3-7year,7-20year,20-40year and40-60year. By the pre-experiments of virus titer and serum titer, the virus titers were100TCID50and the serum dilution ratio is1:8. Then Micro-serum neutralization (SN) assay were used to analysis serum neutralizations of Adenovirus with positive control, normal cell control, serum toxicity control. The results were assessed according the standards:it is negative if with100%CPE by the sample, and it is positive with less than50%CPE.The SN assay results showed that the positive rates of neutralizing antibodies of Adv3, Adv4and Adv7increased with age except0-6month group. It was the lowest in Adv3neutralizing antibodies positive rate in the0-12month group and1-3year group. In the20-40year and40-60year group, the Adv3antibody positive was the highest at78%.; The antibody positive rate of Adv4was the lowest in the6-12month group and the higher in the7-20year group(60%). The same positive rate was in the20-40year group, and the highest rate was82%in the40-60year group; the lowest positive rate of Adv7was only5%in the1-3year group, then, the positive rate was increased with age. In the40-60year group, the positive rate was the highest (76%).The samples of0-1year group were analyzed according the divided group:0-1month,1-3month,3-6month and6-12month. The results showed the highest antibody positive rate of Adv3was58%in the0-1month group. Then the rate decreased to the lowest positive rate13%in the3-6month group. The trends of the positive rate of Adv7were same to Adv3, although Adv7positive rate for each group was lower than the rate of Adv3. According to the chi-square analysis, χ2=12.05, P <0.01between Adv3and Adv7, their antibody positive rates were of a significant difference. The trends of the positive rate of Adv4antibody were different with Adv3and Adv7. The positive rate was also the highest in the0-1month group, but in the other groups, the positive rate was maintained to10-13%.Type-specific neutralizing antibodies were induced by different serotype adenovirus in vivo. One serum sample could possess several different types of neutralizing antibodies. According the data of three kinds of antibodies, the proportion without any neutralizing antibody against HAdV-3,-4and-7was the highest in the6-12month group and1-3year group, which was more than70%. The proportion decreased from54%to9%in the3-7year group with age. While in the40-60age group population, none was antibody negative. The positive rate with only one kind of adenovirus antibody was highest in the0-6month group and7-20year group. The positive rate with two or three kinds of adenovirus antibodies increased with age. In the40-60year group, their neutralizing antibody positive rate is42%and46%, respectively.Comparing the antibody positive rate of the group people of different environment, the results showed that:the antibody positive rate of people who work in hospital was slightly higher than the other, but by the chi-square test, x2=0.19, P> 0.05, there was no significant difference between the two sets of data.In this part, we compared these data from several different contents. The results showed the antibody positive rate increased with age. In the6months to3years old people, neutralizing antibody positive rate was lower than the others. The periods could be the best time to vaccination. The prevalent trends of Adv3antibody was likely to Adv7, while the prevalent of Adv4antibody was characteristic. The results showed that adenovirus infection was widespread in the Guangzhou. In the40-60year group, everyone has been infected by adenovirus once or more. These researches would be helpful to vaccine and vector development.Part II Preparation and identification of monoclonal antibodies against Adenovirus type3In this study, the Adv3purified by sucrose density gradient centrifugation was used to immunize BALB/c mice. The immunized mouse spleen cells were fused with the SP2/0cells by PEG3350. Then, the fused cells were selected by HAT medium and cloned by dilution culture. The monoclonal antibody titer was measured by indirect ELISA method. At last, a hybridoma cell strain1B6was obtained and cryopreserved. The immunoglobulin subtype of monoclonal antibody1B6was identified as IgGl subtype, the light chain of antibody was κ chain. Then ascites antibodies were purified by ammonium persulfate-caprylic acid precipitation for high titer. By ELISA and Western blotting, the1B6MAb was specific to Adv3virus particles and the hexon protein from prokaryotic expression. By immunocytochemistry, the MAb could recognize the cells infected by Adv3. So the1B6MAb might to a linear natural epitope.In the part, we obtained a stable hybridoma cell lines, which was identified by hexon protein of prokaryotic expression and the natural virus. The MAb is likely to complement to a linear natural hexon epitope. Work is needed to be further confirmed whether this epitope could to be a neutralizing effect.PartⅢ Identification of neutralizing epitopes of Adenovirus type3The recombinant virus of Ad3EGF which was inserted by EGFP and A7HAd3egf whose hexon of Ad3EGF was substituted by the hexon of Adv7was replication-competent.In vitro Adv neutralization experiments,1B6MAb was mixed with the two recombinant viruses and incubated for1h. Then, the mixtures were transferred to96-well plates containing85-95%confluent monolayers of HEp-2cells. After72h, infected cells were analyzed with a Varioskan Flash Multimode Reader (Thermo Scientific) to measure the EGFP expression. Results showed the virus replication were decreased in the1:64antibody dilution because the relative fluorescent value of Ad3EGF and A7HAd3egf viruses are more than70. When the antibody was diluted to1:32, replication of Ad3EGF was inhibited by the1B6antibody for the fluorescence value dropped to40. In the1:8antibody dilutions, relative fluorescence value has been less than20, which indicated the virus replication was inhibited significantly. The relative fluorescence value of virus A7HAd3egf was stabilized at80-100, when the replication could not be inhibited. The results of in vitro Adv neutralization experiments showed that the1B6antibody was a neutralizing antibody, and was specific to the hexon protein of Adv3.By blasting the amino acid sequence of Adv3-gz01and Adv7-GZ8adenovirus hexon, six HVRs were expressed in prokaryotic systems. The results showed1B6MAb was identical to R7and R7s. R7s specificity was lower than the R7which could be caused for several amino acid residues absently.To indentify the epitopes, we designed a competitive inhibition ELISA assay. The data showed that the R7fragment epitope can be completely closed to the MAb1B6, while the R7s fragment closed function is weak, which only partially enclosed the MAb.Finally, some Adv vectors which had been constructed by colleagues of the group identified with the MAb by ELISA. Results showed MAb1B6could specifically identify the vectors which had the hexon of Adv3. As R7epitope could be indentified in the natural structure, it is a continuous linear natural epitope, which epitope was consistent with the epitope expressed in prokaryotic systems.In the part,1B6MAb were identified as a neutralization antibody by vitro Adv neutralization experiments. The R7epitope of Adv3hexon also was identified as a continuous linear natural epitopes by competitive ELISA.Conclusioni. The prevalence of neutralizing antibody results show that adult population with Adv antibodies was more than70%. In the40-60years group, none was antibody negative. In the0-6month, more than50%of infants were protected by maternal antibodies. The population of6months to3years was likely to adenovirus infection and vaccination for low antibody positive rate.ii. MAb1B6was a high specificity, high sensitivity neutralizing antibody to the hexon of Adv3.iii. R7epitope was a linear natural neutralizing epitopes of Adv3hexon which can be characteristics to MAb1B6.