*2.3.1. Innate immunity*

Adenoviruses are known to induce robust innate immune responses in their hosts. The adenovirus binds to its receptor(s) (such as Coxsackie adenovirus receptor or CAR, integrin αvβ5 heparin sulfate proteoglycans, CD46, sialic acid, etc.) on host cells and gains entry into the cytoplasm [28–32]. However, phagocytic antigen-presenting cells such as macrophages and dendritic cells can also take up virus particles through scavenger receptors [33]. Inside a host cell, the virus can be recognized by various intracellular molecular sensors such as Toll-like receptors (TLRs), RIG-I like receptors (RLRs), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), cytosolic DNA sensors, and effector molecules [34–36].

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 cytokine IL-12 [41].

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

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

Adenoviruses are known to induce robust innate immune responses in their hosts. The adenovirus binds to its receptor(s) (such as Coxsackie adenovirus receptor or CAR, integrin αvβ5 heparin sulfate proteoglycans, CD46, sialic acid, etc.) on host cells and gains entry into the cytoplasm [28–32]. However, phagocytic antigen-presenting cells such as macrophages and dendritic cells can also take up virus particles through scavenger receptors [33]. Inside a host cell, the virus can be recognized by various intracellular molecular sensors such as Toll-like receptors (TLRs), RIG-I like receptors (RLRs), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), cytosolic DNA sensors, and effector

designing of adenoviral vectors.

56 Adenoviruses

**2.3. Immunity to adenoviruses**

*2.3.1. Innate immunity*

molecules [34–36].

immune responses to Adenoviruses in detail.

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

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

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 domain responsible for ATP-dependent self-oligomerization, a C-terminal leucine-rich repeat (LRR) domain that senses the presence of a ligand, and a variable N-terminal interaction domain that mediates protein-protein interactions. The NLR activation leads to the formation of inflammasomes with the help of microtubules [58]. The human adenovirus activates the formation of two types of inflammasomes in myeloid cells: absent in melanoma-2 (AIM2) and NLR-pyrin domain (PYD)-containing protein (NALP3). The activation of inflammasomes induces inflammatory responses via NF-κB signaling, converts pro-IL1β and pro-IL18 into IL1β and IL18, respectively, and can lead to DNA fragmentation, membrane pore formation, and eventually cell death by pyroptosis [59].

administrations that can be done and reduce the efficiency of transgene expression. Both humoral and cellular immune responses are discussed in detail in the following sections.

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

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

59

The surface antigens of adenovirus, penton, hexon, and fiber proteins are involved in host cell receptor interaction and can be neutralized by antibodies. The impact of neutralizing antibodies (nAbs) on adenoviral gene and antigen delivery has been studied extensively [80–82]. The passive transfer of serum from Ad immune mice or purified nAbs against adenoviruses decreases the vector transgene expression and induction of transgene-specific cellular and humoral responses. Depletion of antibodies against fiber, penton, and hexon by affinity chromatography has been shown to significantly enhance the transgene expression and induced immune responses. Furthermore, hexon-specific antibodies seem to play a relatively dominant role *in vivo* in comparison to other antigens. The hexon-specific nAbs are directed against exposed hypervariable loop-containing regions (HVR) on the surface of the virus particle. The pre-existing immunity to a prevalent human adenovirus 5 (HAd5) can be overcome by replacing the entire HAd5 hexon sequence of exposed epitopes with the HVR from a different serotype [6, 82–84]. Consequently, three amino acid substitutions in one of the HVRs significantly reduced neutralization by polyclonal serum raised against a chimpanzee Ad serotype 68 (ChAd68)-derived vector [83, 85]. Conversely, replacing SAd24 hexon with SAd23 into an SAd24/Pan7 vector resulted in reduced transgene expression in mice with pre-existing SAd23/Pan6 immunity [86]. Additionally, nAb against Ad fiber has a minimal effect *in vivo* as shown in a mouse study where nAbs induced by HAd7 administration weakly neutralized HAd7 fiber-expressing chimeric HAd5 vector, indicating that other non-fiber capsid protein-specific nAbs also have a major role in neutralization. Although these *in vivo* animal studies shed light on the relative importance of pre-existing fiber-specific nAbs, they do not accurately reflect the impact of preexisting immunity on adenovector efficiency in humans. In most of these animal studies, the pre-existing fiberspecific nAbs were induced by a single administration of Ad, which induced nAbs with poor breadth and at far lower levels than in humans exposed to repeated natural Ad infections. The nAbs against other surface antigens such as penton may also work against Ad in a synergistic fashion along with fiber-specific nAbs. Together, these factors contribute to more effective neutralization of Ad-based vectors and result in poor transgene expression and induced transgene antigen-specific immunity in humans. Importantly, Ad-specific nAbs are mostly serotype-specific and have very limited to no neutralization capability of other serotypes. This serotype-specificity is due to high sequence heterogeneity of epitopes in the hexon HVR and fiber knob among different Ad serotypes. Moreover, the nAbs are not the only factor in pre-existing Ad immunity; non-neutralizing Abs can also hamper Ad vector efficacy via Fc receptor-dependent cytotoxicity, complement-mediated lysis, and opsonization. In humans, Ad infections are very common and nearly everyone contains some levels of Ad-specific antibodies. The high seroprevalence of Ad-specific antibodies is a major roadblock in adenoviral vector development, and strategies to circumvent these

*2.3.2.1. Humoral immunity*

must be examined.

The early adenoviral proteins E1, E3, and E4 interfere with the innate immune signaling and help in evading host immune responses. The E1A protein allows hijacking of cell cycle, inducing apoptosis, evading immuning, inducing tumorigenesis, and expressing viral genes [60, 61]. The E1A has been reported to block type 1 IFN-inducible gene expression [62–64], type 2 IFN-γ-dependent HLA class II expression, and IFN-β expression in response to double-stranded RNA due to inhibition of transcription complex formation [62]. Furthermore, E1A inhibits IFN-alpha-stimulated transcription factor 3 (ISGF3), IFN-stimulated genes (ISGs) [65, 66], and immunoproteasomes, resulting in reduced antigen presentation to T cells [67]. In addition, the early adenoviral proteins E1B-19K and E1B-55K antagonize p53-mediated apoptosis [68, 69], and E1B-55K interferes with the induction of IFN-inducible genes [70, 71]. The E1B-55K and E4 proteins induce proteasome-mediated degradation of defense factor death domain-associated protein (Daxx) resulting in the removal of viral transcription blocking allowing viral gene expression [72, 73]. E1B-55K and E4 protein complex also result in inhibition of antiviral innate immune responses [74–77]. The E3 protein has several immune modulatory functions. It blocks the surface transport of MHC-class I molecule and also reduces NK cell receptors on host cells, masking infected cells from detection by immune cells [78]. Furthermore, the E3 protein also inhibits apoptosis of adenovirusinfected cells by downregulation of death receptors [79].

The induction of innate immune responses is critical in adenoviral vector-based strategies. On the one hand, the gene transfer vector should have minimal activation of innate immune signaling to allow efficient gene delivery without immune activation. On the other hand, adenoviral vector-based vaccine antigen delivery could benefit from adenovirus's intrinsic property of innate immune activation that results in efficient activation of transgene-specific adaptive immune responses. Therefore, careful engineering of adenoviral vectors can serve the purpose of both gene and vaccine antigen delivery.

### *2.3.2. Adaptive immunity*

The adaptive immune responses to adenoviruses are directed against both early and late viral proteins. They include both neutralizing antibodies and T cells against viral surface antigens such as hexon, penton, and fiber proteins. However, these adaptive immune responses against adenoviral antigens also present major obstacles in adenoviral vector development as gene delivery, and vaccine antigen carriers limit the number of administrations that can be done and reduce the efficiency of transgene expression. Both humoral and cellular immune responses are discussed in detail in the following sections.

### *2.3.2.1. Humoral immunity*

domain responsible for ATP-dependent self-oligomerization, a C-terminal leucine-rich repeat (LRR) domain that senses the presence of a ligand, and a variable N-terminal interaction domain that mediates protein-protein interactions. The NLR activation leads to the formation of inflammasomes with the help of microtubules [58]. The human adenovirus activates the formation of two types of inflammasomes in myeloid cells: absent in melanoma-2 (AIM2) and NLR-pyrin domain (PYD)-containing protein (NALP3). The activation of inflammasomes induces inflammatory responses via NF-κB signaling, converts pro-IL1β and pro-IL18 into IL1β and IL18, respectively, and can lead to DNA fragmentation, membrane pore formation,

The early adenoviral proteins E1, E3, and E4 interfere with the innate immune signaling and help in evading host immune responses. The E1A protein allows hijacking of cell cycle, inducing apoptosis, evading immuning, inducing tumorigenesis, and expressing viral genes [60, 61]. The E1A has been reported to block type 1 IFN-inducible gene expression [62–64], type 2 IFN-γ-dependent HLA class II expression, and IFN-β expression in response to double-stranded RNA due to inhibition of transcription complex formation [62]. Furthermore, E1A inhibits IFN-alpha-stimulated transcription factor 3 (ISGF3), IFN-stimulated genes (ISGs) [65, 66], and immunoproteasomes, resulting in reduced antigen presentation to T cells [67]. In addition, the early adenoviral proteins E1B-19K and E1B-55K antagonize p53-mediated apoptosis [68, 69], and E1B-55K interferes with the induction of IFN-inducible genes [70, 71]. The E1B-55K and E4 proteins induce proteasome-mediated degradation of defense factor death domain-associated protein (Daxx) resulting in the removal of viral transcription blocking allowing viral gene expression [72, 73]. E1B-55K and E4 protein complex also result in inhibition of antiviral innate immune responses [74–77]. The E3 protein has several immune modulatory functions. It blocks the surface transport of MHC-class I molecule and also reduces NK cell receptors on host cells, masking infected cells from detection by immune cells [78]. Furthermore, the E3 protein also inhibits apoptosis of adenovirus-

The induction of innate immune responses is critical in adenoviral vector-based strategies. On the one hand, the gene transfer vector should have minimal activation of innate immune signaling to allow efficient gene delivery without immune activation. On the other hand, adenoviral vector-based vaccine antigen delivery could benefit from adenovirus's intrinsic property of innate immune activation that results in efficient activation of transgene-specific adaptive immune responses. Therefore, careful engineering of adenoviral vectors can serve

The adaptive immune responses to adenoviruses are directed against both early and late viral proteins. They include both neutralizing antibodies and T cells against viral surface antigens such as hexon, penton, and fiber proteins. However, these adaptive immune responses against adenoviral antigens also present major obstacles in adenoviral vector development as gene delivery, and vaccine antigen carriers limit the number of

and eventually cell death by pyroptosis [59].

58 Adenoviruses

infected cells by downregulation of death receptors [79].

the purpose of both gene and vaccine antigen delivery.

*2.3.2. Adaptive immunity*

The surface antigens of adenovirus, penton, hexon, and fiber proteins are involved in host cell receptor interaction and can be neutralized by antibodies. The impact of neutralizing antibodies (nAbs) on adenoviral gene and antigen delivery has been studied extensively [80–82]. The passive transfer of serum from Ad immune mice or purified nAbs against adenoviruses decreases the vector transgene expression and induction of transgene-specific cellular and humoral responses. Depletion of antibodies against fiber, penton, and hexon by affinity chromatography has been shown to significantly enhance the transgene expression and induced immune responses. Furthermore, hexon-specific antibodies seem to play a relatively dominant role *in vivo* in comparison to other antigens. The hexon-specific nAbs are directed against exposed hypervariable loop-containing regions (HVR) on the surface of the virus particle. The pre-existing immunity to a prevalent human adenovirus 5 (HAd5) can be overcome by replacing the entire HAd5 hexon sequence of exposed epitopes with the HVR from a different serotype [6, 82–84]. Consequently, three amino acid substitutions in one of the HVRs significantly reduced neutralization by polyclonal serum raised against a chimpanzee Ad serotype 68 (ChAd68)-derived vector [83, 85]. Conversely, replacing SAd24 hexon with SAd23 into an SAd24/Pan7 vector resulted in reduced transgene expression in mice with pre-existing SAd23/Pan6 immunity [86]. Additionally, nAb against Ad fiber has a minimal effect *in vivo* as shown in a mouse study where nAbs induced by HAd7 administration weakly neutralized HAd7 fiber-expressing chimeric HAd5 vector, indicating that other non-fiber capsid protein-specific nAbs also have a major role in neutralization. Although these *in vivo* animal studies shed light on the relative importance of pre-existing fiber-specific nAbs, they do not accurately reflect the impact of preexisting immunity on adenovector efficiency in humans. In most of these animal studies, the pre-existing fiberspecific nAbs were induced by a single administration of Ad, which induced nAbs with poor breadth and at far lower levels than in humans exposed to repeated natural Ad infections. The nAbs against other surface antigens such as penton may also work against Ad in a synergistic fashion along with fiber-specific nAbs. Together, these factors contribute to more effective neutralization of Ad-based vectors and result in poor transgene expression and induced transgene antigen-specific immunity in humans. Importantly, Ad-specific nAbs are mostly serotype-specific and have very limited to no neutralization capability of other serotypes. This serotype-specificity is due to high sequence heterogeneity of epitopes in the hexon HVR and fiber knob among different Ad serotypes. Moreover, the nAbs are not the only factor in pre-existing Ad immunity; non-neutralizing Abs can also hamper Ad vector efficacy via Fc receptor-dependent cytotoxicity, complement-mediated lysis, and opsonization. In humans, Ad infections are very common and nearly everyone contains some levels of Ad-specific antibodies. The high seroprevalence of Ad-specific antibodies is a major roadblock in adenoviral vector development, and strategies to circumvent these must be examined.

### *2.3.2.2. Cellular immunity*

In humans, Ad vector-specific CD4<sup>+</sup> Th1 cells have been detected, but the frequencies of these cells decrease with age [87]. The CD8<sup>+</sup> T cell responses to different structural proteins have 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, both human and mouse-derived CD4+ and CD8+ T cells cross react with human and simian Ad 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 infection.

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

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

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

61

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

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,

major concern in the second-generation adenoviral vectors [110] (**Figure 2A–C**).

manner [104, 105] (**Figure 2A–C**).

*3.1.2. Second generation*

*3.1.3. Third generation*
