Isolation of novel simian adenoviruses from macaques for development of a vector for human gene therapy and vaccines

Abstract

Both human and non-human simian adenoviruses (HAdVs and SAdVs, respectively) have been used as gene therapy and vaccine vectors. The high prevalence of HAdVs and the neutralizing antibodies associated with prior infection, may limit HAdV-based vector use in human subjects. To overcome this drawback, a vector derived from a newly isolated and characterized macaque adenovirus was constructed. SAdVs (33.9%) were screened from 115 SAdV fecal samples collected at a zoological park. One novel SAdV was isolated and the whole genome was sequenced and analyzed. The pre-existing neutralizing antibody levels were very low against this isolate (10%). Interestingly, SAdV vector constructs that lack E3 region could not produce infectious progeny in HEK293 cells, suggesting that the E3 region is necessary for SAdV replication. The absence of E3 region could be compensated for by replacement with HAdV-5 E4orf6; the resultant construct could replicate well in HEK293 cells. The enhanced Green Fluorescent Protein (eGFP) was inserted into SAdV E3 region and expressed at high level. One-step growth curve showed that the replication of the SAdVs with HAdV-5 E4orf6 substitution and E1/E3 deletion was similar to that of wild-type SAdVs in HEK293 cells, but the modified SAdVs were replication-deficient in A549 cells which lack HAdV-5 E1A and E1B. Finally, we demonstrated that GZ3-12 could infect cells expressing hCAR or hDSG2 receptors. The successful isolation, characterization, and modification of novel SAdVs provide a potentially important vaccine and gene therapy candidate and a new strategy for the rapid acquisition and development of non-HAdV-based alternative vectors for human health applications. IMPORTANCE Adenoviruses are widely used in gene therapy and vaccine delivery. Due to the high prevalence of human adenoviruses (HAdVs), the pre-existing immunity against HAdVs in humans is common, which limits the wide and repetitive use of HAdV vectors. In contrast, the pre-existing immunity against simian adenoviruses (SAdVs) is low in humans. Therefore, we performed epidemiological investigations of SAdVs in simians and found that the SAdV prevalence was as high as 33.9%. The whole-genome sequencing and sequence analysis showed SAdV diversity and possible cross species transmission. One isolate with low level of pre-existing neutralizing antibodies in humans was used to construct replication-deficient SAdV vectors with E4orf6 substitution and E1/E3 deletion. Interestingly, we found that the E3 region plays a critical role in its replication in human cells, but the absence of this region could be compensated for by the E4orf6 from HAdV-5 and the E1 expression intrinsic to HEK293 cells.

A modified recombinant adenovirus vector containing dual rabies virus G expression cassettes confers robust and long-lasting humoral immunity in mice, cats, and dogs

“…Low biocontainment requirements for production; v. Ability to promote durable and robust humoral and cellular immune response [ 41 ]. Researchers have developed several novel adenoviral vectors for gene delivery, such as simian adenoviruses [ 26 ], human adenovirus type 4 (Ad4) [ 42 ], human adenovirus type 35 [ 43 ] (Ad35) vectors, and chimpanzee adenovirus type 68 (cAd68) [ 44 ]. Previous studies have demonstrated that these novel adenovirus vectors mediate the diverse immune responses in hosts [ 45 ].…”

“…In 2021, the recombinant COVID-19 vaccine (Adenovirus Type 5 vector) was approved by the National Medical Products Administration (NMPA) for SARS-COV-2 prevention [ 24 ]. However, pre-existing Ad5-neutralizing antibodies in the population have somewhat hampered its prospects as a human vaccine vector [ 26 ]. Adenoviruses are strictly species-specific, and animals do not naturally become infected with human adenoviruses.…”