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A large number of gastrointestinal, acute respiratory and eye infections in humans are caused by adenoviruses. Adenoviruses have a wide host range from mice, monkeys to humans. Adenoviruses were first isolated in human adenoids (tonsils), from which the name is derived. Adenoviruses represent the largest nonenveloped viruses, because they are the maximum size able to be transported through the endosome (envelope fusion is not necessary). The virion also has a unique "spike" or fiber associated with each penton base of the capsid that aids in attachment to the host cell through the coxsackie-adenovirus receptor on the surface of the host cell (Figure 1).
Figure 1. Adenoviral particle organisation.
Adenoviruses are medium-sized (90–100 nm), nonenveloped (naked) icosahedral viruses composed of a nucleocapsid and a double-stranded linear DNA genome. There are more than 51 different serotypes in humans, which are responsible for 5–10% of upper respiratory infections in children, and a number of infections in adults as well. Adenoviruses are primarily spread via respiratory droplets. However, they can also be spread by fecal routes as well. Adenovirus infections often show up as tonsilitis, conjunctivitis, an ear infection, or croup. Adenoviruses can also cause gastroenteritis. A combination of conjunctivitis and tonsilitis is particularly common with adenovirus infections.
Life Cycle of Adenovirus
The virus life cycle initiates with attachment to cell surface receptors. (1) Attachment: The virus attaches to the cell surface receptors through the interaction of the pentons with alphaVbeta3 and alphaVbeta5 integrin proteins. (2) Internalization: It is subsequently internalized by receptor-mediated endocytosis, escapes from endosome to the cytosol and translocates to the nucleus to begin viral transcription and replication. (3) Replication: Cell death is triggered to release viral progeny upon completion of the viral life cycle. Transcriptional units are conventionally referred to as early (E1a, E1b, E2, E3, and E4), delayed early (proteins IX and IVa2) or late genes (L1-L5) based on the temporal expression relative to the onset of viral DNA replication. The early gene products are commonly involved in gene transcription, replication, host immune suppression and apoptosis inhibition of host cell, and late gene products are responsible for virion assembly.
Adenovirus vectors are likely the most frequently used viral vectors, mainly due to the high levels of transgene expression that can be obtained in a broad range of host cells. Adenovirus vectors have many advantages: Adenoviruses are well studied and adenovirus vectors can be grown into high titer stable stocks, they infect non-dividing and dividing cells of different types, and they are maintained in cells as an episome. The vectors are predominantly nonintegrating, nonenveloped, episomal, double-stranded DNA viruses.
Most adenovirus vectors are genetically modified versions of the serotype Ad5. Two types of vectors are available: replication-defective (RD) and replication-competent (RC). Replication-defective vectors do not carry the virus E1A gene. E1A initiates the program of viral gene transcription and reprograms multiple aspects of cell function and behavior. These E1A-deleted vectors are usually constructed from plasmids or virus DNA containing the genetically modified genome; the vectors are grown on complementing cell lines which retain and express the E1A gene. For oncolytic gene therapy for cancer, where replication-competent viral gene expression is needed, E1A has been either mutated or placed under tumor-specific transcriptional control. Today’s vectors also have deletions of various E2 and E4 genes since viral proteins encoded by these DNA sequences were shown to induce most of the host immune response. Apart from decreased toxicity, there is the advantage of prolonged gene expression in vivo.
Adenoviral Vectors for Vaccines and Cancer Therapy
Because adenoviruses are immunogenic and there is much preexisting immunity to the virus, their vectors are not optimal for long-term complementation of faulty genes in monogenic diseases, but rather for the delivery of vaccines and cancer therapy. RD adenovirus vectors have been employed extensively as vaccines because they induce a strong humoral and especially a strong T cell response, tending to a T helper 1 type response, to the transgene expressed by the vector. RC oncolytic vectors have been engineered to replicate preferentially in cancer cells, leading to apoptosis, direct cell death, or increasing the sensitivity of cancer cells to antitumor drugs. The vectors have proven highly safe and effective against gliomas.
Figure 2. Using adenoviral vectors to deliver antigens.
Advexin is an E1-minus E3-minus RD Ad5 vector expressing p53 from the CMV promoter in the deleted E1 region. At least a dozen clinical trials have been conducted with Advexin at multiple testing sites for cancers including head and neck squamous cell carcinoma (HNSCC), hepatocellular carcinoma (HCC), non-small cell lung cancer, colorectal cancer, prostate cancer, ovary cancer, breast cancer, bladder cancer, and glioma. In phase I and II trials on HNSCC, the vector was injected as a monotherapy directly into tumors at doses up to 2.5 × 1011 vp/dose. The treatment was judged to be safe. Then, Advexin was evaluated in a phase III trial for advanced recurrent HNSCC that compared Advexin as monotherapy vs. Advexin in combination with methotrexate. Again, the vector was well tolerated, and evidence of anti-tumor activity was obtained.
In summary, a number of clinical trials and clinical studies with oncolytic adenovirus vectors have shown that these vectors have anti-tumor activity and are very well tolerated. Given the large number of patients that have been treated, there have been only a few somewhat serious adverse events (e.g. grade 3 ileus), but even those resolved in a quite short period of time. Moreover, in nearly all the clinical studies, the presence of vector, usually in the serum, was detected and quantitated by qPCR. This method can detect a fragment of DNA, not necessarily complete genomes or, more importantly, infectious virus. Thus, it is possible that the amount of infectious vector present in the blood of patients in these trials is less than is suggested by the qPCR data. If so, then these vectors may be even safer than we may think.