Xiao-Hui Zou

Systemic and mucosal immunity in mice elicited by a single immunization with human adenovirus type 5 or 41 vector‐based vaccines carrying the spike protein of Middle East respiratory syndrome coronavirus

An ideal vaccine against mucosal pathogens such as Middle East respiratory syndrome coronavirus (MERS‐CoV) should confer sustained, protective immunity at both systemic and mucosal levels. Here, we evaluated the in vivo systemic and mucosal antigen‐specific immune responses induced by a single intramuscular or intragastric administration of recombinant adenoviral type 5 (Ad5) or type 41 (Ad41) ‐based vaccines expressing the MERS‐CoV spike (S) protein. Intragastric administration of either Ad5‐S or Ad41‐S induced antigen‐specific IgG and neutralizing antibody in serum; however, antigen‐specific T‐cell responses were not detected. In contrast, after a single intramuscular dose of Ad5‐S or Ad41‐S, functional antigen‐specific T‐cell responses were elicited in the spleen and pulmonary lymphocytes of the mice, which persisted for several months. Both rAd‐based vaccines administered intramuscularly induced systemic humoral immune responses (neutralizing IgG antibodies). Our results show that a single dose of Ad5‐S‐ or Ad41‐S‐based vaccines represents an appealing strategy for the control of MERS‐CoV infection and transmission.

Human adenovirus type 41 possesses different amount of short and long fibers in the virion

To determine the ratio of short fiber (sfiber) to long fiber (lfiber) in human adenovirus type 41 (HAdV-41) virions, sfiber and lfiber were expressed in E. coli, quantified, and used as loading standards in Western blot. Densitometric analyses of the standard and target bands indicated that the ratio of sfiber to lfiber in virions was 5.7±0.7. Sfiber-deleted HAdV-41, HAdV-41-DSF-GFP, was constructed, and Western blot analysis showed that the amount of lfiber in HAdV-41-DSF-GFP was about 7.3±1.9 times of that in HAdV-41 virions, confirming a ratio of approximate 6 for sfiber to lfiber in HAdV-41. In HAdV-41-infected cells, mRNAs of the sfiber and lfiber were comparable in quantity, while the expression at protein level was significantly different. Our results suggested an unequal number of short and long fibers, which might result from their differential protein expression during HAdV-41 packaging. The method used here could be extended to quantify other trace proteins.

An improved HAdV-41 E1B55K-expressing 293 cell line for packaging fastidious adenovirus

Human adenovirus type 41 (HAdV-41) is difficult to cultivate under laboratory conditions. Tripartite leader sequence (TPL) of HAdV-41 was cloned, inserted into eukaryotic expression plasmid, and used to establish a HAdV-41 E1B55K-transduced cell line (293TE7). HAdV-41 E1B55K was expressed more abundantly in 293TE7 than in 293E12, an HAdV-41 E1B55K-expressing cell line developed previously. After being infected with E1-deleted HAdV-41 vector (HAdV-41-GFP), 293TE7 synthesized more viral genomic DNA and structural proteins, which led ultimately to a significant increase of the yield of progeny viruses. Typically, 293TE7 produced progeny viruses 3-15 times more than 293E12 did, depending on the amount of seed viruses and culture time. These data demonstrated that 293TE7 was an effective packaging cell line, and implied its application for wild-type HAdV-41 isolation, HAdV-41 virological study and recombinant HAdV-41 construction.

Inefficient export of viral late mRNA contributes to fastidiousness of human adenovirus type 41 (HAdV-41) in 293 cells.

The human adenovirus (HAdV) early protein E1B55K interacts with E4orf6 to form an E3 ubiquitin ligase complex, which plays key roles in virus replication. To illustrate the reason for the fastidiousness of HAdV-41 in 293 cells, interaction between heterotypic E1B55K and E4orf6 proteins was investigated. HAdV-5 E1B55K could interact with HAdV-41 E4orf6, and vice versa. To form E1B55K/E4orf6 E3 ubiquitin ligase, HAdV-41 E4orf6 recruited Cul2 while HAdV-5 E4orf6 interacted with Cul5. The ligase complex formed by HAdV-5 E1B55K and HAdV-41 E4orf6 could cause the degradation of p53 and Mre11. However, in E1-deleted HAdV-41-infected 293TE7 cells, which expressed HAdV-41 E1B55K, viral late mRNAs were exported from nucleus more efficiently and accumulated to a higher concentration in cytoplasm when compared with that in infected 293 cells. These results suggested that interaction between homotypic E1B55K and E4orf6 was indispensable for efficient export of viral late mRNAs.

Enhanced growth of recombinant human adenovirus type 41 (HAdV-41) carrying ADP gene.

Human adenovirus type 41 (HAdV-41) has the potential to be constructed as a gene transfer vector for oral vaccine or gene therapy targeting gastrointestinal tract. Block in release of progeny virus from host cell severely affects the yield during virus amplification. In this study, HAdV-5 adenovirus death protein (ADP) gene was used to replace the open reading frames (ORFs) of the HAdV-41 E3 region to construct a backbone plasmid pAdbone41ADP. Recombinant adenoviral plasmids harboring ADP and GFP genes (pAd41ADP-GFP) were generated. Plaques were formed and HAdV-41-ADP-GFP virus was rescued after transfecting pAd41ADP-GFP into the packaging cell line 293TE32. When amplified on 293TE32 cells, HAdV-41-ADP-GFP virus released to the culture medium was 10-50 times more than control virus HAdV-41-GFP, which did not carry ADP gene. The results demonstrated that incorporation of the ADP gene substantially increased the yield of recombinant HAdV-41 virus through enhancing spread of progeny virus among packaging cells.

Prevalence of serum neutralizing antibodies to adenovirus type 5 (Ad5) and 41 (Ad41) in children is associated with age and sanitary conditions

Abstract Neutralizing antibody (NAb) can dampen the immunogenicity of adenovirus (Ad) vector-based vaccine. Vector systems based on human adenovirus type 41 (Ad41) have been constructed and used to develop recombinant vaccines. Here, we attempted to study the seroprevalence of NAbs to Ad5 and Ad41 among children and adults in Qinghai province, China. The positive rates (titer⩾40) of Ad5 and Ad41 NAb in adults from Xining city were 75.7% and 94.7%, respectively. The moderate/high-positive rates (titer⩾160) of NAb were quite close between the two viruses in adults (70.4% for Ad5 and 73.5% for Ad41). Age-dependent increase of NAb seroprevalence was observed for both viruses in children. NAb-positive rate of Ad41 reached 50% at 3.3–4.6years of age for children from Chengxi district, Xining city, approximately 1.5years earlier than that of Ad5 did. Interestingly, NAb level was also associated with sanitary conditions among young children. For Ad5, 8–15% children (0.2–3.0years of age) from city or town, where the sanitations were relatively better, had moderate/high-positive NAb, while the same rate was 62% for children from villages. For Ad41, 22% children from city, 47% from town and 88% from villages possessed moderate/high-positive NAb. The possible influence of NAb titer distributions on the application of Ad41-vectored vaccines was discussed in detail. Our results suggested that children from places with poor sanitations should be included for comprehensive Ad NAb seroprevalence studies, and provided insights to the applications of Ad41 vectors.

DNA assembly technique simplifies the construction of infectious clone of fowl adenovirus

Plasmid bearing adenovirus genome is generally constructed with the method of homologous recombination in E. coli BJ5183 strain. Here, we utilized Gibson gene assembly technique to generate infectious clone of fowl adenovirus 4 (FAdV-4). Primers flanked with partial inverted terminal repeat (ITR) sequence of FAdV-4 were synthesized to amplify a plasmid backbone containing kanamycin-resistant gene and pBR322 origin (KAN-ORI). DNA assembly was carried out by combining the KAN-ORI fragment, virus genomic DNA and DNA assembly master mix. E. coli competent cells were transformed with the assembled product, and plasmids (pKFAV4) were extracted and confirmed to contain viral genome by restriction analysis and sequencing. Virus was successfully rescued from linear pKFAV4-transfected chicken LMH cells. This approach was further verified in cloning of human adenovirus 5 genome. Our results indicated that DNA assembly technique simplified the construction of infectious clone of adenovirus, suggesting its possible application in virus traditional or reverse genetics.