**2. Adenoviruses**

### **2.1. Genome and proteins**

Adenoviruses contain a 26–45 kb size double-stranded DNA genome, inside their icosahedral virion [1]. The DNA genome of adenoviruses contains two inverted terminal repeats with 100–140 bp flanks on both the ends. Due to its small genome size, adenoviruses employ several strategies to maximally utilize its genome. For example, they encode proteins from both DNA strands, employ alternate-splicing, and use different poly A modifications of its mRNA. Adenoviral genes can be divided into five early and five late genes. Once internalized into target cells, the adenoviruses express the early genes E1A, E1B, E2, E3, and E4, which modulate host gene expression required for adenovirus protein synthesis and replication. The late transcriptional units include L1–L5 and are required in the assembly, release, and lysis of host cells [1, 5, 6] (**Figure 1**).

host cells, E1A stimulates apoptosis by both p53-dependent and -independent pathways [9]. In contrast, the E1B protein inhibits apoptosis by binding to several host cell proteins such as p53, Bak, and BAX proteins [8]. In non-replicating adenoviral vectors, the E1 gene is deleted to render them replication-defective so that it can infect the host cells but cannot multiply. However, for production of non-replicating adenoviral vectors, E1 transfected cells such as HEK293 and

**Figure 1.** Adenovirus structure and genome organization. (A) Graphical representation of adenovirus structure and various proteins. (B) Adenovirus genome organization showing various early (E) and late (L) transcripts and proteins encoded by each transcript. Regions indicated by red with (\*) are deleted in various adenoviral vectors. E1 and E3 regions were deleted in first generation and E1, E2, E3, and/or E4 were deleted in second-generation adenoviral vectors. Most recent adenoviral vectors called helper-dependent adenoviral vectors only contain ITRs and packaging signals.

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Adenoviruses are grouped under the family Adenoviridae, which is divided into five genera: Mastadenovirus, Aviadenovirus, Siadenovirus, Atadenovirus, and Ichtadenovirus. Human adenoviruses, along with many animal adenoviruses (monkeys, cattle, sheep, swine, dogs), belong to the genus Mastadenovirus. Human adenoviruses (HAd) are classified into seven subgroups: A–G and further in to 67 serotypes based on serological properties. The classification of serotypes into subgroups is based on their similarities in genome organization and DNA sequences, host tropism, carcinogenic potential in rodents, and growth properties in cell cultures. Adenoviral serotyping is based on viral surface antigen neutralizing antibodies and by phylogenetic distance (>10%) in the viral genes that encode viral protease, the protein

The genus Aviadenovirus contains bird adenoviruses, while other genera Siadenovirus, Atadenovirus, and Ichtadenovirus contain other adenoviruses of mammals, birds, reptiles, and fishes [13–15]. The adenoviruses isolated from sheep, cattle, deer, possum, and some birds differ from the adenoviruses of the genus Mastadenovirus and are classified under

PER.C6 are used to allow production of replication-defective adenoviral vector [9].

pVIII, the hexon protein, and the DNA polymerase [10–12].

**2.2. Types of adenoviruses**

Figure is adapted from Ref. [209].

Structurally, adenovirus consists of a core of capsid and genome. The viral capsid consists of structural proteins hexon, penton, fiber, IIIa, VIII, and IX. Hexons are major surface structural proteins consisting of 270 trimers, which are arranged as 12 pentamers of pentons at the top of 12 icosahedral vertices. Hexons also contain several hypervariable regions and are the main targets of neutralizing antibodies. In adenoviral vectors, these sites can be engineered to carry vaccine antigen. Each icosahedral vertex gives rise to protruding fibers consisting of 12 trimers. Both penton and fiber proteins serve as ligands for host cell receptors and help in viral entry. The IIIa proteins are located in the inner surface of the capsid and help in the assembly and stabilization of vertex regions and also in the assembly of packaged viral genome. The VI proteins link the outer capsid shell to the inner icosahedral shell. The VIII proteins help in bonding hexons together and are critical for the stability of the viral capsid. The proteins V, VII, and X are associated with the DNA genome and make up the virion core. Terminal protein binds to each end of the DNA genome [6–8] (**Figure 1**).

The early gene first transcribes E1A protein, an essential protein for viral replication. The E1A protein activates the transcription of other viral genes responsible for viral DNA synthesis. In

Adenoviral Vector-Based Vaccines and Gene Therapies: Current Status and Future Prospects http://dx.doi.org/10.5772/intechopen.79697 55

**Figure 1.** Adenovirus structure and genome organization. (A) Graphical representation of adenovirus structure and various proteins. (B) Adenovirus genome organization showing various early (E) and late (L) transcripts and proteins encoded by each transcript. Regions indicated by red with (\*) are deleted in various adenoviral vectors. E1 and E3 regions were deleted in first generation and E1, E2, E3, and/or E4 were deleted in second-generation adenoviral vectors. Most recent adenoviral vectors called helper-dependent adenoviral vectors only contain ITRs and packaging signals. Figure is adapted from Ref. [209].

host cells, E1A stimulates apoptosis by both p53-dependent and -independent pathways [9]. In contrast, the E1B protein inhibits apoptosis by binding to several host cell proteins such as p53, Bak, and BAX proteins [8]. In non-replicating adenoviral vectors, the E1 gene is deleted to render them replication-defective so that it can infect the host cells but cannot multiply. However, for production of non-replicating adenoviral vectors, E1 transfected cells such as HEK293 and PER.C6 are used to allow production of replication-defective adenoviral vector [9].

### **2.2. Types of adenoviruses**

Rowe and his colleagues [2]. Adenoviruses usually cause non-symptomatic respiratory tract infections in both human and animals but can be life-threatening to immunocompromised individuals. Certain human adenovirus serotypes are ubiquitous in children, and most adults carry neutralizing antibodies to adenoviruses [3]. Nonetheless, since their initial use in gene therapy, they have gained wide recognition as a vaccine antigen delivery vehicle and have proven to be safe and efficient vaccine vectors for eliciting protective immune responses against transgene antigens in many animal and human studies. Recently, adenovirus vectors have been employed to attack cancer cells in cancer therapy [4]. In this chapter, we introduce different adenoviruses and their biology and potential for use in gene delivery, vaccine, and therapeutics in several human diseases. In addition, we will discuss their limitations and

Adenoviruses contain a 26–45 kb size double-stranded DNA genome, inside their icosahedral virion [1]. The DNA genome of adenoviruses contains two inverted terminal repeats with 100–140 bp flanks on both the ends. Due to its small genome size, adenoviruses employ several strategies to maximally utilize its genome. For example, they encode proteins from both DNA strands, employ alternate-splicing, and use different poly A modifications of its mRNA. Adenoviral genes can be divided into five early and five late genes. Once internalized into target cells, the adenoviruses express the early genes E1A, E1B, E2, E3, and E4, which modulate host gene expression required for adenovirus protein synthesis and replication. The late transcriptional units include L1–L5 and are required in the assembly, release, and lysis of

Structurally, adenovirus consists of a core of capsid and genome. The viral capsid consists of structural proteins hexon, penton, fiber, IIIa, VIII, and IX. Hexons are major surface structural proteins consisting of 270 trimers, which are arranged as 12 pentamers of pentons at the top of 12 icosahedral vertices. Hexons also contain several hypervariable regions and are the main targets of neutralizing antibodies. In adenoviral vectors, these sites can be engineered to carry vaccine antigen. Each icosahedral vertex gives rise to protruding fibers consisting of 12 trimers. Both penton and fiber proteins serve as ligands for host cell receptors and help in viral entry. The IIIa proteins are located in the inner surface of the capsid and help in the assembly and stabilization of vertex regions and also in the assembly of packaged viral genome. The VI proteins link the outer capsid shell to the inner icosahedral shell. The VIII proteins help in bonding hexons together and are critical for the stability of the viral capsid. The proteins V, VII, and X are associated with the DNA genome and make up the virion core. Terminal protein binds to each end of the DNA

The early gene first transcribes E1A protein, an essential protein for viral replication. The E1A protein activates the transcription of other viral genes responsible for viral DNA synthesis. In

future prospects.

54 Adenoviruses

**2. Adenoviruses**

**2.1. Genome and proteins**

host cells [1, 5, 6] (**Figure 1**).

genome [6–8] (**Figure 1**).

Adenoviruses are grouped under the family Adenoviridae, which is divided into five genera: Mastadenovirus, Aviadenovirus, Siadenovirus, Atadenovirus, and Ichtadenovirus. Human adenoviruses, along with many animal adenoviruses (monkeys, cattle, sheep, swine, dogs), belong to the genus Mastadenovirus. Human adenoviruses (HAd) are classified into seven subgroups: A–G and further in to 67 serotypes based on serological properties. The classification of serotypes into subgroups is based on their similarities in genome organization and DNA sequences, host tropism, carcinogenic potential in rodents, and growth properties in cell cultures. Adenoviral serotyping is based on viral surface antigen neutralizing antibodies and by phylogenetic distance (>10%) in the viral genes that encode viral protease, the protein pVIII, the hexon protein, and the DNA polymerase [10–12].

The genus Aviadenovirus contains bird adenoviruses, while other genera Siadenovirus, Atadenovirus, and Ichtadenovirus contain other adenoviruses of mammals, birds, reptiles, and fishes [13–15]. The adenoviruses isolated from sheep, cattle, deer, possum, and some birds differ from the adenoviruses of the genus Mastadenovirus and are classified under the genus Atadenovirus [6, 16, 17]. The adenoviruses of the genus Mastadenovirus have high A + T (adenine and thymidine)-rich genomes and lack the early region 1 (E1) transcriptional unit. Adenoviruses isolated from many invertebrates are classified under the new genus Siadenovirus. Human and animal adenovirus infections are very common, and the majority of the population of host species contain neutralizing antibodies against the most prevalent serotypes of adenoviruses. Both human and non-human adenoviruses have been studied extensively and are the basis of adenoviral vector-based vaccine and gene therapies [18, 19]. In humans, infection by non-human adenovirus serotypes is not common. However, due to broad tissue tropism and structural and genomic similarity with human adenoviruses, non-human adenoviruses can infect various human tissue types. These properties of adenoviruses encouraged researchers to use non-human adenoviruses as gene or vaccine antigen delivery vectors to mitigate the pre-existing neutralizing immunity that commonly exists against human adenoviral vectors. Several non-human adenoviruses such as bovine Ad serotype 3 (BAd3); canine Ad serotype 2 (CAd2); chimpanzee Ad serotypes 1, 2, 3, 5, 6, 7 and 68 (ChAd1, ChAd2, ChAd3, ChAd5, ChAd6, ChAd7, ChAd68); ovine Ad serotype 7 (OAd7); porcine Ad serotype 3 and 5 (PAd3, PAd5); and fowl Ad serotypes 1, 8, 9, and 10 (FAd1, FAd8, FAd9, FAd10) are currently being tested as vaccine or gene delivery vectors [18, 20–22]. Extensive research in the molecular biology of both human and non-human Ads has helped in better understanding of the adenoviruses and designing of adenoviral vectors.

Cytokines such as IL-8 and TNF-α enhance the entry of human adenovirus type C by increasing the availability of CAR and integrin receptors, which facilitate the adenovirus to enter through clathrin-mediated dynamin-dependent endocytosis [27, 33, 37, 38]. The type B human adenoviruses use CD46 or desmoglein-2 and enter host cells through macropinocytosis [39, 40]; this also results in the suppression of IFN-γ-induced production of proinflammatory

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One of the major drawbacks of the use of adenovirus in gene therapy is the induction of undesired innate immune responses. In liver and spleen, the resident macrophages can sense and trap blood-borne adenovirus and induce inflammatory response mediators [42, 43]. Adenovirus also activates TLR2-dependent expression of chemokines such as MCP-1 and RANTES. In mice, TLR2 deficiency resulted in reduced NF-κB activation and humoral responses to HAd vector antigens and transgene-encoded antigens [42]. However, TLR2 deficiency did not result in complete inhibition of acute and adaptive responses to HAd, suggesting the involvement of an additional pathway [44]. The cellular β3 integrins were recently reported to interact with arginine-glycine-aspartic acid (RGD) motifs of viral homo-pentameric penton base protein during viral entry, which results in the processing of inactive IL1α into active cytokine in a MyD88-, TRIF-, and TRAF6-independent signaling pathway [43]. The IL1α plays a major role in adenovirus-induced inflammatory responses. The IL1R-deficient mice or wild-type mice treated with anti-IL-1 antibodies demonstrated reduced inflammatory responses as well as hepatotoxicity in adenovirus infection [45]. Further, the interaction between the adenoviral RGD motif and host β3 integrin mediates chemokine secretion, leukocyte infiltration, as well

TLR9 also plays a significant role in innate immunity against adenoviruses. Macrophages have been reported to sense adenovirus, helper-dependent adenoviral vector and recombinant E1 and E3-deleted adenovirus through TLR9 [47, 48]. The TLR9-deficient mice show reduced proinflammatory responses and IFN-α production upon adenoviral vector delivery. In a mouse model pf keratitis, adenovirus induced TLR9-dependent IL6 production and monocyte infiltration of the cornea; however, chemokine secretion and keratitis development were TLR9-independent [49, 50]. Another study showed that recombinant adenovirus-induced type I IFN production in plasmacytoid dendritic cells (pDCs) is TLR9-MyD88-dependent but

The viral DNA also plays a critical role in the induction of innate immune responses as empty adenoviral particles are found to be poor inducers of innate responses [51]. The presence of double-stranded RNA with 5′-triphosphate groups in the cytoplasm of target cells is sensed by cytosolic PRR such as RIG-I, and viral DNA and RNA are recognized by intracellular PRRs such as TLR3, 7, and 8 present on the endosomal membrane [48, 52–55]. The double-stranded DNA is sensed by TLR9 in the intracellular environment and also by DNA-dependent activator of IRFs (DAI), DNA-dependent protein kinase (DNA-PK), IFN-γ-inducible protein 16 (IFI16), DEAD (Asp-Glu-Ala-Asp) box polypeptide 41 (DDX41), and by cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) [34, 35, 56, 57]. Other cytosolic viral DNA sensors are NOD-like receptors (NLRs), which consist of a central nucleotide-binding

as corneal inflammation in human adenovirus serotype 37 infections [46].

in myeloid DCs (mDCs) and macrophages, it is TLR9-independent [48].

cytokine IL-12 [41].

### **2.3. Immunity to adenoviruses**

Initially, a host detects the invading virus by sensing unique pathogen-associated molecular patterns (PAMPs) present on the pathogen through pattern recognition receptors (PRRs). Once activated, these PRRs transmit signal to express type I interferons (IFNs) and proinflammatory cytokines which inhibit viral replication and recruit various innate immune cells to the site of infection [23–27]. These initial events ensure the efficient activation and presentation of viral antigens by the antigen-presenting cells to T cells and result in the induction of adaptive immune responses. In the following sections, we will discuss innate and adaptive immune responses to Adenoviruses in detail.
