**3.1. Construction of adenoviral vectors**

Adenoviruses are engineered to make them safe and efficient for human use as vaccine and gene and cancer therapy vectors by deleting certain genome sequences. Initially, human adenoviruses, especially adenovirus 5 (HAd5), were developed as gene delivery vectors. Since the first generation of adenoviral vectors, based on E1 deletion, a number of different strategies have been employed to further improve the gene-carrying capacity and safety by deleting more genes. These strategies can be summarized under following three categories.

### *3.1.1. First generation*

First-generation adenoviral vectors were prepared by deleting early gene E1 to render them replication-defective and create space for a transgene sequence of up to 4.5 kb [92]. Since these vectors lack the E1 region essential for their replication, cell lines such as human embryonic kidney cells (HEK293) were engineered to incorporate viral E1 region [93]. The E1 region in the HEK293 cell line provides trans-complementation and allows the replication of the adenoviral vector [94–97]. These adenoviral vectors carry native tissue transduction capability and efficiently express the transgene in target host cells. However, there are possibilities of spontaneous homologous recombination between vector and E1 regions during amplification inside HEK293 cells, which might enable replication competent adenoviral (RCA) vectors to emerge [98]. To mitigate this problem, another cell line, human embryonic retinoblasts (PERC.6), was made by inserting an expression cassette for the adenoviral E1 region with its own promoter (ubiquitous phosphoglycokinase, PGK) [99]. This eliminates the adenoviral vector homologous regions from the E1 promoter and therefore the chances of recombination [100]. Adenoviral E3 region proteins are known to inhibit immunological pathways [101]. Therefore, the adenoviral E3 region was removed either partially or completely without affecting *in vivo* viral amplification [102]. These deletions in E1 and E3 regions allowed insertion of even larger cargo sequences (up to 8 kb) of two independent genes [103]. Due to the absence of the E1 region, adenoviral vectors are not able to transcribe other early and late viral proteins, although host cellular factors enable these proteins to be expressed at very low levels. This low-level expression of viral protein and subsequent presentation on the cell surface by MHC class I molecules induce robust cytotoxic T cell immune responses. Deletion of E1 is additionally beneficial since adenoviral proteins have toxic effects and induce cell death in a dose-dependent manner [104, 105] (**Figure 2A–C**).

### *3.1.2. Second generation*

*2.3.2.2. Cellular immunity*

60 Adenoviruses

infection.

**3. Adenoviral vectors**

*3.1.1. First generation*

**3.1. Construction of adenoviral vectors**

In humans, Ad vector-specific CD4<sup>+</sup>

cells decrease with age [87]. The CD8<sup>+</sup>

both human and mouse-derived CD4+

Th1 cells have been detected, but the frequencies of these

also been detected in animals in response to adenovirus infection or adenovector administration [19, 80, 88]. Due to extensive homology between different adenoviral structural antigens,

serotypes [20, 89, 90]. Similar to nAb, pre-existing Ad-specific T cells can also reduce Ad vector transgene expression and immunity. Furthermore, Ad-specific T cells have been detected in 80–100% of human subjects in various studies, which make them even more important in Ad vector development [89]. The human studies examining both nAbs and T cells demonstrated a higher proportion of individuals possessing T cell responses compared to nAbs against Ad. The pre-existing Ad-specific T cells have greater consequences for Ad vaccine vector development due to their cross-reactive nature, higher distribution in the human population, and their multifunctional nature [22]. Finally, human Ad vector has also recently been reported to induce cross-reactive hepatitis C virus-specific humoral and cellular immune responses [91]. Widespread use of adenoviral vectors in humans will induce such cross-reactive immune responses at high levels, which might be beneficial or detrimental in the development of natural immunity against HCV and affect the immunopathology and disease progression of HCV

Adenoviruses are engineered to make them safe and efficient for human use as vaccine and gene and cancer therapy vectors by deleting certain genome sequences. Initially, human adenoviruses, especially adenovirus 5 (HAd5), were developed as gene delivery vectors. Since the first generation of adenoviral vectors, based on E1 deletion, a number of different strategies have been employed to further improve the gene-carrying capacity and safety by deleting more genes. These strategies can be summarized under following three categories.

First-generation adenoviral vectors were prepared by deleting early gene E1 to render them replication-defective and create space for a transgene sequence of up to 4.5 kb [92]. Since these vectors lack the E1 region essential for their replication, cell lines such as human embryonic kidney cells (HEK293) were engineered to incorporate viral E1 region [93]. The E1 region in the HEK293 cell line provides trans-complementation and allows the replication of the adenoviral vector [94–97]. These adenoviral vectors carry native tissue transduction capability and efficiently express the transgene in target host cells. However, there are possibilities of spontaneous homologous recombination between vector and E1

and CD8+

T cell responses to different structural proteins have

T cells cross react with human and simian Ad

Second-generation adenoviral vectors possess deletions in E2 or E4 regions that encode for proteins required for replication in target cells [106–108]. These deleted proteins were complemented in trans by cell lines (such as HEK293) to allow for vector propagation. These second-generation vectors provided additional space for larger cargo sequences (10.5 kb) with up to four independent expression cassettes and eliminated the possibility of generating replication-competent adenoviruses during amplification. This deletion of early viral genes impacts the amplification of viral vector in cell culture and results in lower yields due to inefficient complementation by the producer cell lines [107, 109]. These vectors also have been reported to have lower transgene expression. Immunogenicity and cellular toxicity are still a major concern in the second-generation adenoviral vectors [110] (**Figure 2A–C**).

### *3.1.3. Third generation*

Third-generation adenoviral vectors are also called "high capacity adenoviral vectors" (HCAds) because they can accept cargo sequences up to 36 Kb [111–113]. The HCAds were generated by deleting all viral sequences except the ITRs and the packaging signal [114]. For replication of third-generation adenovirus vectors in cell culture, instead of the complementation by the viral genes encoded by host cells, an additional adenoviral helper virus is provided. Therefore, the third-generation adenoviral vectors are also called helperdependent or "gutless" adenoviral vectors [115–117]. The helper adenovirus is generated like a first-generation adenoviral vector and includes packaging signal flanking loxP sites. The vector is produced in HEK293 cells that constitutively express Cre recombinase by simultaneously transducing helper virus and the HCAd genome. This allows the synthesis of adenoviral proteins by the helper virus and enables assembly of viral capsids,

efficiently transduces host cells due to reduced induction of anti-adenoviral neutralizing antibodies [118, 122]. The HCAds can simultaneously encode multiple transgene cassettes. Although the HCAds provide a much superior vector system, they are more complicated to generate compared to previous generations of adenoviral vectors and also have possibility of helper virus contamination due to inefficient Cre-mediated excision of the helper virus

Adenoviral Vector-Based Vaccines and Gene Therapies: Current Status and Future Prospects

http://dx.doi.org/10.5772/intechopen.79697

63

Since their first use in gene therapy, adenoviral vectors have progressed significantly and are currently being tested clinically in several gene therapy, vaccine vector, and anticancer

Adenoviruses have a unique ability to infect a broad range of cell types. Therefore, adenovirus-based vectors can be used to transduce and deliver transgenes to different cell types including both replicating and quiescent cell populations. This property of adenoviral vectors is extremely important in gene therapy and puts adenoviral vectors on top of viral vectors for gene delivery. Furthermore, adenovirus vectors do not integrate into host genomes but stay as episomal DNA in the nucleus of host cells. Modern adenoviral vectors can take multiple gene cassettes, up to 36 kb of foreign DNA, which make them suitable for delivering virtually any size of gene. In 1992, for the first time, a first-generation adenoviral vector was used to deliver and express alpha-1 antitrypsin (A1AT) in hepatocytes of a patient who had alpha-1 antitrypsin deficiency [124]. In another study, an E1–E3 deleted HAd5 adenoviral vector was used to deliver an A1AT gene to lung tissues [125]. Later, using adenoviral vectors, a number of attempts were made to deliver dysfunctional or deficient genes, which were responsible for several human genetic diseases and conditions. Cystic fibrosis is one such human genetic disease, in which the gene CFTR (cystic fibrosis transmembrane conductance regulator) becomes dysfunctional due to mutation. Adenoviral vector was used to deliver CFTR genes to lung tissues [126]. In another study, the adenoviral vector was used to deliver the gene for ornithine transcarbamylase, which is required in the urea cycle and is responsible for ornithine-transcarbamylase deficiency [127, 128]. These studies faced several challenges including humoral and cellular immunity to adenoviral vectors upon repeated administration of vector, cellular cytotoxicity, and oncogenesis [129]. These trials raised serious safety concerns for using adenoviral vectors in gene therapy and resulted in a sharp decline in their use. The reasons for these problems were studied extensively and addressed by constructing new adenoviral vectors. The adenoviral immunogenicity and cytotoxicity were suspected to be due to lowlevel expression of several viral proteins. The newer generations of adenoviral vectors had these adenoviral genes removed, and hence the vector immunogenicity and toxicity were significantly reduced. The new generations of adenoviral vectors have raised new hope in adenoviral vector-based gene delivery. Currently, a number of gene therapy clinical trials

packaging signal [123] (**Figure 2D**).

studies.

*3.2.1. Gene therapy*

**3.2. Current applications of adenoviral vectors**

**Figure 2.** Methods of preparation of different types of adenoviral vectors. (A) First generation. The target gene is cloned into a shuttle vector containing 5′-ITR, a packaging signal, and the sequence for homologous recombination. This shuttle vector and an adenoviral backbone vector are transfected into HEK-293 cells, and adenoviral vector is created through homologous recombination between the two vectors. (B) First or second generation. The target gene is cloned into a shuttle vector that contains 5′-ITR, a packaging signal, and an LoxP site(s). This shuttle vector and a LoxP-containing adenoviral backbone vector are joined together through Cre recombinase-mediated recombination either *in vitro* or in HEK-293 cells. (C) First or second generation. The target gene is cloned into a shuttle vector containing 5′-ITR, a packaging signal, and a kanamycin-containing bacterial replication sequence flanked with two homologous arms. The homologous recombination between the linearized shuttle vector and ampicillin-resistant adenoviral backbone vector takes place in bacterial cells (BJ5183), and adenoviral plasmids are selected on kanamycin. This plasmid is linearized and transfected in HEK-293 cells for adenoviral vector production. (D) Third generation. The target gene is cloned into a transfer vector that only contains ITRs and a packaging signal. A helper adenovirus is used to generate the adenoviral vector. Modified HEK-293 cells are used for adenoviral production, which prevent packaging of helper adenovirus due to deletion of packaging signal. Figure is adapted from Ref. [209].

resulting in the packaging of HCAd genome only. The helper virus genome-packaging signal is excised by Cre-mediated recombination of the loxP sites, thus preventing helper virus genomes from assembling into viral particles. In some production systems, other recombinases like *Saccharomyces cerevisiae*-derived Flp recombinase [118] or bacteriophage-derived phiC31 integrase [119] have also been used. Third-generation vectors have several benefits over first- and second-generation adenoviral vectors. These include less cellular toxicity and reduced immunogenicity [120, 121], thereby providing a flexible vector system that efficiently transduces host cells due to reduced induction of anti-adenoviral neutralizing antibodies [118, 122]. The HCAds can simultaneously encode multiple transgene cassettes. Although the HCAds provide a much superior vector system, they are more complicated to generate compared to previous generations of adenoviral vectors and also have possibility of helper virus contamination due to inefficient Cre-mediated excision of the helper virus packaging signal [123] (**Figure 2D**).

### **3.2. Current applications of adenoviral vectors**

Since their first use in gene therapy, adenoviral vectors have progressed significantly and are currently being tested clinically in several gene therapy, vaccine vector, and anticancer studies.
