Zhuo-Zhuang Lu

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.

Novel recombinant adenovirus type 41 vector and its biological properties

Human adenovirus serotype 41 (Ad41) is a natural pathogen of the digestive tract and can cause gastroenteritis. There has been interest in reconstructing Ad41 as a gene delivery vector targeting the gastrointestinal tract, which is hampered by its fastidiousness.

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.

A three-plasmid system for construction of armed oncolytic adenovirus

There is growing interest in the use of oncolytic virus as a tool in cancer gene therapy. However, construction of oncolytic adenovirus (Ad) is not an easy task due to lack of convenient, robust methods. A three-plasmid system was introduced for construction of armed oncolytic Ad. Besides the pShuttle-CMV and pAdEasy-1, a third plasmid (pTE-ME1), harboring the E1 region of Ad5, was generated and included in this system. In pTE-ME1, the promoter of E1A was deleted and replaced with a multiple-cloning site (MCS). A therapeutic gene and tissue-specific promoter (TSP) could be inserted routinely into the MCS of pShuttle-CMV and pTE-ME1, respectively. The modified E1 region could then be excised from pTE-ME1 and integrated into the therapeutic gene-containing pShuttle-CMV to form the final shuttle plasmid. This shuttle plasmid was recombined with pAdEasy-1 in Escherichia coli strain BJ5183 to generate Ad plasmid. Finally, the oncolytic Ad could be rescued in Ad plasmid-transfected packaging cells. The GFP gene and the promoter of telomerase reverse transcriptase (TERTp) were chosen as the transgene and TSP, respectively, to test this system. Two oncolytic Ads, Ad-GFP-TPE and Ad-GFP-D19K, were generated successfully. Their oncolytic and replicating abilities were investigated in TERT-positive tumor cells. The results suggest that the three-plasmid system was practicable and could be used to construct other transcriptionally regulated oncolytic Ads carrying a therapeutic gene.

Construction of an infectious clone of human adenovirus type 41

Human adenovirus type 41 (HAdV-41) is well known for its fastidiousness in cell culture. To construct an infectious clone of HAdV-41, a DNA fragment containing the left and right ends of HAdV-41 as well as a kanamycin resistance gene and a pBR322 replication origin was excised from the previously constructed plasmid pAd41-GFP. Using homologous recombination, the plasmid pKAd41 was generated by co-transformation of the E. coli BJ5183 strain with this fragment and HAdV-41 genomic DNA. Virus was rescued from pKAd41-transfected 293TE7 cells, a HAdV-41 E1B55K–expressing cell line. The genomic integrity of the rescued virus was verified by restriction analysis and sequencing. Two fibers on the virion were confirmed by western blot. Immunofluorescence showed that more expression of the hexon protein could be found in 293TE7 cells than in 293 cells after HAdV-41 infection. The feature of non-lytic replication was preserved in 293TE7 cells, since very few progeny HAdV-41 viruses were released to the culture medium. These results show that pKAd41 is an effective infectious clone and suggest that the combination of pKAd41 and 293TE7 cells is an ideal system for virological study of HAdV-41.

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.

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.