A pShuttle plasmid psAd3H containing the complete hexon gene, which contains the epsilon epitope of HAdV-3, was constructed. This construct also contained the left and right homologous regions that enable homologous recombination with pAdEasy to produce infectious clone prAd3H (Fig. 1). The resultant plasmid was confirmed by DNA sequencing, which not only provided confirmation of the complete hexon gene sequence but also verified that no mutations were introduced. Prior to sequencing, as a rapid check, restriction endonuclease analysis (REA) was performed to characterize the recombinant plasmid rapidly. Restriction maps generated with five restriction endonucleases were consistent with the in silico restriction maps predicted by the Vector NTI 11.5.1 software (Invitrogen Corp; San Diego, CA, USA) (Zhao et al. 2014; Yu et al. 2016; Zhang et al. 2017; Pan et al. 2018) (Fig. 2).
Recombinant plasmid prAd3H was linearized by Pac I and transfected into AD293 cells to rescue the recombinant adenovirus rAd3H. Green fluorescence was observed under fluorescence microscopy and the fluorescence density was noted to increase each subsequent day, an indication that the recombinant adenovirus was replicating. At day 10 post-transfection, the virus culture was frozen and thawed three times; after centrifugation, the supernatant was inoculated into AD293 cells. Green fluorescence and CPE were observed and recorded at day 8 post-infection (Fig. 3). The increased fluorescence and CPE suggested that the recombinant virus rAd3H was rescued successfully from AD293 cells.
The transcription of the rAd3H hexon gene was confirmed by RT-PCR. A rAd5 virus that containing adenovirus type 5 genome except for E1 and E3 regions was used as a positive control. Viral RNA was extracted from a rAd3H culture and digested with DNase. After digestion, the presumed viral RNA was used as a PCR template for amplifying the hexon gene as a check for any residual DNA. No PCR products were found from these samples, which indicated that the extracted RNA did not contain viral genomic DNA (Fig. 4A). After reverse transcription, the cDNA was used as a PCR template to validate the insert. The resultant amplification of the hexon gene confirmed the rAd3H recombinant adenoviruses. (Fig. 4B).
Figure 4. RT-PCR analysis of rAd3H hexon transcription (A, B) and Western blot confirmation of the HAdV-3 hexon protein expression from rAd3H (C). A RNA as template for PCR. Lanes: (M) DL10000 marker; (1) rAd3H; (2) rAd5; (3) mock; (4) PCR positive control; (5) PCR negative control; B cDNA as template for PCR. (M) DL10000 marker; (1–2) rAd3H; (3–4) rAd5; (5) mock; (6) PCR positive control; (7) PCR negative control; C Lanes: (1–2) rAd3H cell supernatant; (3) negative control, rAd5 cell supernatant; (4) mock, supernatant of AD293 cells.
Expression of the HAdV-3 hexon protein in the culture supernatant was also detected by Western blots (Fig. 4C), in which HAdV-3 specific mouse monoclonal antibody was used to identify HAdV-3 hexon. The rAd5 adenovirus was used as the negative control, as it contained the HAdV-5 hexon gene but not the HAdV-3 hexon gene. The HAdV-3 hexon protein of 108 kDa was confirmed in the rAd3H recombinant vaccine strain while no band was found in the rAd5 and mock samples, indicating that during the culture, the HAdV-3 hexon protein of rAd3H can be expressed successfully.
The harvested viral culture from rAd3H was negative-stained by sodium phosphor-tungstate and observed under electron microscopy. Typical adenovirus particles, 70–90 nm in diameter, were clearly visible (Fig. 5). This further confirmed the successful infection and replication of the recombinant HAdV-3 vaccine in AD293 cells.
The replication efficiencies of rAd3H, rAd5 and HAdV-3 strain GZ01 were compared by quantification of genomic DNA copies using a real-time PCR method. Primers targeting the conserved region of the penton base gene to represent the genomic DNA number were used in the real-time PCR amplification. The two one-step growth curves of rAd3H and HAdV-3 strain GZ01 showed that the replication efficacy of rAd3H viruses were similar to that of HAdV-3 wild-type strain in AD293 cells, both of which increased similarly during the 60 h post infection. The peaks of DNA copy number of both rAd5 and rAd3H strains were higher than HAdV-3 GZ01 strain, which might be because HAdV-5 genomic DNA is the backbone of both strains and the viruses are cultured in AD293 cells which highly expressed HAdV-5 E1A and E1B genes that promote the HAdV-5 DNA replication (Fig. 6).
Figure 6. Replication dynamics curves of rAd3H bearing HAdV-3 hexon gene, rAd5 and HAdV-3 wild-type strain GZ01. AD293 cells were infected by the two viruses and harvested at 12, 24, 36, 48, 60, 72, 84, 96, 108, and 120 h post infection. Viral genomic DNA copy numbers in cells and supernatants were determined by real-time PCR for the penton base region.
To assess the stability of the insert, recombinant vaccine strain rAd3H was passaged in AD293 cells for at least 20 generations. The virus stability of rAd3H harvested from the 20 generations of culture was verified by PCR amplification and DNA sequencing of the HAdV-3 and HAdV-5 hexon genes. There were no amino acid mutations detected in the HAdV-3 and HAdV-5 hexon gene of rAd3H upon sequence verification. This indicated that both hexon genes in this recombinant vaccine strain are well compatible with each other during the viral replication. As expected, both fluorescence and CPE were observed in the AD293 cells infected by rAd3H from the first to the twentieth generations (Fig. 7A–7D). However, no green fluorescence was detected in the A549 cells infected by rAd3H which was the culture from the first to the twentieth generations of rAd3H in A549 cells (Fig. 7E–7H), which indicated no infectious viral particles produced in A549 cells. PCR amplification of the hexon gene from the virus culture in A549 cells showed no product, which indicated that rAd3H could only replicate in AD293 cells, but not in the A549 cells due to the E1 deletion. No reverse mutations occurred during the continuous passages of rAd3H in AD293 cells. All the results presented confirmed the stability of the vaccine stain rAd3H in AD293 cells (Fig. 7).
Figure 7. Light and fluorescent microscopy observation of cells infected by the recombinant vaccine rAd3H. A, B AD293 cells infected by the 1st generation of rAd3H; C, D AD293 cells infected by the 20th generation of rAd3H; E, F A549 cells infected by the 1st generation of rAd3H cultured in A549 cells; G, H A549 cells infected by the 20th generation of rAd3H cultured in A549 cells. A, C, E, G are in fluorescence vision; B, D, F, H are in white light vision (100 ×) at day 5 post-infection.
Mice were either inoculated with HAdV-3 wild-type strain GZ01 or immunized with the rAd3H recombinant vaccine by either the intranasal route or intramuscular route, respectively, to assess the antibody titer. At day 14 post inoculation/immunization, the mice were boosted with the same inoculant. Mouse sera were collected at day 21, 28, 35, and 42 after the prime inoculation/immunization. The 50% neutralizing antibody titers of mouse sera in the intranasal or intramuscular groups collected at different time points were determined by the microculture neutralization test (Table 1). All of the mice immunized with rAd3H or HAdV-3 GZ01 virus intranasally or intramuscularly produced neutralizing antibodies. However, the mice immunized intramuscularly produced higher and more lasting antibody titers than the intranasally immunized mice. Therefore, apparently the intramuscular immunization was more effective than the intranasal immunization in provoking the immune response. In the wild-type GZ01-innoculated mice, higher neutralizing antibody titers were produced in both intranasal and intramuscular groups, which might be associated with the replication-competence of wild-type adenoviruses as compared with the replication-deficient rAd3H vaccine strain.
Serum collection time rAd3H immunization HAdV-3 wildtype GZ01 inoculation Intranasal Intramuscular Intranasal Intramuscular D21 1:250 1:270 1:1000 1:1428 D28 1:200 1:250 1:1111 1:1000 D35 1:167 1:333 1:1111 1:1667 D42 1:158 1:248 1:916 1:1024 The mice were either immunized with the rAd3H recombinant vaccine or inoculated with HAdV-3 wildtype strain GZ01 intranasally or intramuscularly. At day 21, 28, 35, and 42 after the prime inoculation/immunization, the neutralizing antibodies against HAdV-3 were titrated.
Table 1. Determination of the 50% neutralizing antibody titer in mouse sera by the microculture neutralization test.