Comparison of the Life Cycles of Genetically Distant Species C and Species D Human Adenoviruses Ad6 and Ad26 in Human Cells

Abstract

Our understanding of adenovirus (Ad) biology is largely extrapolated from human species C Ad5. Most humans are immune to Ad5, so lower-seroprevalence viruses like human Ad6 and Ad26 are being tested as therapeutic vectors. Ad6 and Ad26 differ at the DNA level by 34%. To better understand how this might impact their biology, we examined the life cycle of the two viruses in human lung cells in vitro. Both viruses infected A549 cells with similar efficiencies, executed DNA replication with identical kinetics within 12 h, and began killing cells within 72 h. While Ad6-infected cells remained adherent until death, Ad26-infected cells detached within 12 h of infection but remained viable. Next-generation sequencing (NGS) of mRNA from infected cells demonstrated that viral transcripts constituted 1% of cellular mRNAs within 6 h and 8 to 16% within 12 h. Quantitative PCR and NGS revealed the activation of key early genes at 6 h and transition to late gene activation by 12 h by both viruses. There were marked differences in the balance of E1A and E1B activation by the two viruses and in the expression of E3 immune evasion mRNAs. Ad6 was markedly more effective at suppressing major histocompatibility complex class I (MHC I) display on the cell surface and in evading TRAIL-mediated apoptosis than was Ad26. These data demonstrate shared as well as divergent life cycles in these genetically distant human adenoviruses. An understanding of these differences expands the knowledge of alternative Ad species and may inform the selection of related Ads for therapeutic development. IMPORTANCEA burgeoning number of adenoviruses (Ads) are being harnessed as therapeutics, yet the biology of these viruses is generally extrapolated from Ad2 and Ad5. Here, we are the first to compare the transcriptional programs of two genetically distant Ads by mRNA next-generation sequencing (NGS). Species C Ad6 and Ad26 are being pursued as lower-seroprevalence Ad vectors but differ at the DNA level by 34%. Head-to-head comparison in human lung cells by NGS revealed that the two viruses generally conform to our general understanding of the Ad transcriptional program. However, fine mapping revealed subtle and strong differences in how these two viruses execute these programs, including differences in the balance of E1A and E1B mRNAs and in E3 immune evasion genes. This suggests that not all adenoviruses behave like Ad2 and Ad5 and that they may have unique strategies to infect cells and evade the immune system. T here are currently nearly 60 genotypes of adenoviruses (Ads) that infect humans, causing an array of ocular, respiratory, digestive, and systemic infections (reviewed in reference 1). Human mastadenoviruses (HAdVs) fall into genetically grouped species A (HAdV-A) through species G (HAdV-G), with the level of sequence diversity across the virome being as high as 40% (2). Despite this drastic genetic diversity, the biology of most Ads is often extrapolated from the study of just two respiratory serotypes of HAdV-C, Ad2 and Ad5.Transcripti...

Cryo-EM structure of human adenovirus D26 reveals the conservation of structural organization among human adenoviruses

“…Human species-D HAdV-D26, originally isolated from stool samples ( 9 , 10 ), is genetically distant and 34% different at the DNA level from the well-characterized HAdV-C5 virus ( 11 ). A study carried out in 1963 in male inmate “volunteers” demonstrated that ocular inoculation of HAdV-D26 results in conjunctivitis that peaks within 4 days and viral infection in the throat peaking at 10 days, followed by shedding of virus in the stool for as long as 40 days after eye infection ( 10 ).…”

Retargeting adenoviruses for therapeutic applications and vaccines

“…We and others have delved into the biologies of other human and nonhuman Ads in the quest for new functionalities or to evade anti-Ad5 immunity in patients and to have non-Ad5 genetic platforms for vector engineering [23, 38,42,[46][47][48]97,[100][101][102][103][104][105][106][107][108][109][110][111]. They also allow one to avoid pre-existing immune responses against certain Ads (i.e., Ad5) and to vary the Ad serotype between treatments in a shell game called serotype-switching [100,112].…”

“…We also directly compared the genetic activation programs of two of these genetically distant Ads: human species C Ad6 and species D Ad26 [106]. Ad6 Fig.…”

Single-cycle adenovirus vectors in the current vaccine landscape

“…For example, SC-Ad does not replicate in most mouse cells, but will replicate its DNA 500-fold in Ad-permissive hamster cells (16). In contrast, human Ads can replicate their genomes up to 10,000-fold in human cells (102). This suggests that promising preclinical data in animals may under-estimate SC-Ad potency in humans.…”