*3.3.2. Pre-existing adenoviral immunity*

*3.3.1. Tissue tropism and transgene expression*

**Table 3.** Oncolytic therapy: adenoviruses in clinical trial.

11 DNX-2401 with IFNγ E1A Δ24

**S. no.**

70 Adenoviruses

3 HAd5-yCD/

4 ONCOS-102 with cyclophosphamide

10 DNX-2401 with Temozolomide

(IMRT)

12 Ad5-yCD/mutTKSR39rep-ADP with intensitymodulated radiation therapy

5 VCN-01 with or without abraxane and gemcitabine

6 VCN-01 with abraxane and gemcitabine

mutTKSR39rep-hIL12

1 ICOVIR-5 E2F-E1A Δ24

RGD

DM-1-E2F-E1A Δ24 RGD

DM-1-E2F-E1A Δ24 RGD

9 Colo-Ad1 Ad11p/Ad3 — Colon, non-small

E1A Δ24 RGD

RGD

13 OBP-301 hTERT — Hepatocellular

7 CG0070 E2F-E1A Granulocyte

Adenoviruses can infect a diverse range of mammalian cell types. Infection to host cells is mediated by binding of adenoviral fiber protein to host cell surface receptors followed by recruitment of RGD motifs on penton bases to bind the host cell alpha-integrins. Most

**Adenoviral vector (biologic) Modification Transgene Target/condition Phase ClinicalTrials** 

deaminase (CD)/ tyrosine kinase (TK) hIL12

5/3 Δ24 GM-CSF Advanced

macrophage colony-

8 CG0070 E2F-E1A GM-CSF Bladder cancer II/III NCT01438112

E1B-55K CD/TK Prostate

stimulating factor (GM-CSF)

2 LOAd703 5/3 Δ24 CD40L & 4-1BBL Pancreatic cancer I/IIa NCT02705196

E1B-55K Cytosine

**identifier**

I NCT01598129

I NCT02045602

I NCT02045589

I NCT02053220

I NCT01956734

II/III NCT00583492

I/II NCT02293850

— Solid tumors I NCT01864759

neoplasms

pancreatic cancer

cell lung cancer, bladder, renal cancer

multiforme

carcinoma

carcinoma

— Brain tumors I NCT02197169

tumors

Hyaluronidase Advanced solid

Hyaluronidase Advanced

— Glioblastoma

Prostate cancer I NCT02555397

Bladder cancer III NCT02365818

The impact of pre-existing immunity against adenoviral vectors has been discussed in the previous section under adaptive immunity. To avoid the pre-existing immunity against adenoviral vectors, several strategies are being employed as discussed below.

### *3.3.2.1. Use of alternative less frequent adenoviruses for vector*

Several human adenoviruses with low seroprevalence such as HAd2, HAd26, and HAd35 were identified and developed into vectors [186]. The seroprevalence of these rare human adenovirus serotypes is very low, and hence the effect of pre-existing immunity is minimal [81, 187]. HAd26 and HAd35 vectors have been tested in phase I clinical trials and proven to be safe. However, the immunogenicity and efficacy of these low seroprevalence vectors are reported to be lower in comparison to more prevalent HAd5. These results are very concerning and warrant further investigation to find the reasons for the poor performance of these vectors. The nAb and T cells against HAd5 do not cross react with HAd35 but nAbs and T cells against another common serotype, HAd2, cross react with HAd35 and reduce the immunogenicity and efficacy of the HAd35 vectors [188, 189].

To avoid the cross-reactive immunity due to closely related serotypes of Ad, more genetically distant Ad serotypes including animal and bird Ad were developed as viral vectors. Among nonhuman adenovirus vectors, chimpanzee-derived adenovirus vector (ChAd) is the most widely used. In comparison to HAd, the nAbs against ChAd have been found to be less prevalent. For example, nAbs against ChAd7 were detected in only 15% of American, European, Chinese, and African population [186]. Similarly, nAbs against ChAd6 are also low in these populations except Africans, which have about 40% ChAd6-specific nAbs [81, 186]. Several chimpanzee adenoviral vector-based vaccines, such as ChAd7 for Ebola virus, ChAd6 for rabies, and ChAd6, ChAd7, and ChAd9 for malaria, have shown high efficacy in animal models [190]. Furthermore, ChAd63-based malaria and ChAd3-based hepatitis C virus vaccines have shown to be safe and highly immunogenic in phase I clinical trial [191, 192]. Despite low seroprevalence of ChAd vectors in humans, pre-existing cross-reactive T cells against many conserved viral antigens are still a major concern. The HAd-induced ChAd cross-reactive T cells have been reported against ChAd6, ChAd7, ChAd24, ChAd32, and ChAd68 [22, 80, 89, 90]. The negative effects of these cross-reactive T cells on ChAd have been demonstrated in several animal models [22, 89]. The impact of the pre-existing cross-reactive T cells could be far greater in the clinical setting where humans are repeatedly exposed to various serotypes of adenovirus and carry a far broader diversity and a higher frequency of cross-reactive T cells.

responses compared to single vaccination or homologous prime-boost immunizations. The cellular immune responses induced by DNA prime and HAd boost were not affected by pre-existing HAd5 immunity. These findings were further confirmed in a clinical trial. Another preclinical study involving *Plasmodium* or SARS antigens encoded by Modified Vaccinia Ankara (MVA)/adenoviral vector as prime/boost showed induction of robust T cell and Ab responses of higher magnitude compared to Ad/DNA regimens. Finally, a ChAd63/ MVA prime-boost strategy is now being evaluated in clinical trials as a vaccine against malaria, HIV, and HCV [191, 202–204]. The heterologous prime/boost strategy seems very effective in circumventing the pre-existing immunity against adenoviral vectors in studies conducted so far; however, the vehicle for priming and/or boosting must be carefully

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73

Adenoviral antigens induce robust antibody and T cell immune responses. Recent studies have shown that adenoviral-derived epitopes can dominate over the transgene-derived epitopes and hinder the induction of transgene-specific immunity. This impairment of transgene-specific immune responses in naive vaccinees is due to immune competition. Epitopes derived from an adenovirus vector were shown to inhibit the induction of HIV

hexon486-494 epitope. The IL2, however, could not restore GagL85-93 responsiveness in Ad-based immunization, likely due to the presence of other epitopes in the Ad vector [206]. Another study demonstrated that plasmid DNA, but not adenovirus vector-encoding hepatitis B surface antigen (HBsAg), primed CD8 T cells against subdominant HBsAg epitopes [207]. These studies suggest that adenoviral antigen-specific T-cell immunity is primed efficiently during adenoviral vector-based immunization, which can limit the immunogenicity of adenoviral vector-encoded transgenic antigens. These studies highlight the need for modifications of the vector or the transgene used in immunization to circumvent or dominate over the adenoviral vector-specific epitopes and induce more effective transgene-

We discovered an unusual and interesting phenomenon that non-recombinant HAd5 vector induces robust cross-reactive immune responses toward hepatitis C virus (HCV) antigens [91]. Upon further investigation, we found that adenoviral proteins contain extensive homologies with various peptide epitopes derived from HCV antigens. These observations led us to investigate the adenoviral vector-induced HCV cross-reactive immune responses in detail in both mice and humans. In mice, we demonstrated that Ad vector alone can induce potent, broad anti-HCV cross-reactive immunity that can significantly reduce viral load upon challenge with infectious chimeric Vaccinia-HCV. Furthermore, we also detected

T cells [205]. This study demonstrated that competition occurs at

T cells, and co-immunization with an interleukin 2-encoding

T cell responses in the presence of an adenoviral

designed and selected.

GagL85-93-specific CD8<sup>+</sup>

specific immunity.

the level of responding CD8+

plasmid restored GagL85-93-specific CD8<sup>+</sup>

*3.3.4. Heterologous immunity induced by adenoviral antigens*

*3.3.3. Immunodominance over transgene immunity*

Apart from rare human and chimpanzee adenoviruses, several other adenoviruses derived from animals such as bovine, porcine, ovine, canine, and fowl are also being explored for vector development [151, 193–195]. The human population lack nAbs against these adenoviruses, and therefore, the vectors derived from these adenoviruses could be more efficacious in comparison to HAd and ChAd. The mouse models with experimentally induced pre-existing immunity by the administration of HAd vector demonstrated a lack of nAbs and CD4+ T cells against PAd3 and BAd3 [18]. Furthermore, BAd3- or PAd3-based influenza virus vaccine demonstrated high efficacy even in the presence of pre-existing HAd5 immunity. There was also no effect of pre-existing HAd5 immunity on transgene expression, immunogenicity, and efficacy in animal models. However, their potential benefits still need to be proven in humans.

### *3.3.2.2. Routes of immunization*

Several studies have reported that different routes of immunizations can negate the detrimental effect of pre-existing immunity. This outcome could be at least partly due to evasion of tissue-resident Ad-specific T cells when using different routes of immunization. Tissueresident CD8 memory T cells remain confined to a specific tissue. As such, they are not systemic and do not prevent Ad vector infection in distant tissues. These tissue-resident T cells are induced by tissue-derived migratory dendritic cells during priming, which activate T cells with specific tissue-homing molecules [196–198]. In a non-human primate model, HAd5 induced protective immune responses by intranasal/intratracheal immunization were not affected by pre-existing HAd5 immunity that had been induced by intramuscular administration of an unrelated HAd5 vector [199]. Although this strategy has shown promising results in animal models, it has not been verified in humans. This strategy is however restricted by limited routes feasible for administration in humans. Moreover, the nAbs induced by adenoviral infection or adenoviral vector-based vaccination can be detected in any part of the body, which may affect the transgene expression and immune responses irrespective of the alternative route used for subsequent adenovector administration. However, nAbs are generally present in blood and are in most cases not cross-reactive to different serotypes or subtypes and Ads from different host species [200].

### *3.3.2.3. Heterologous prime-boost strategy*

Another strategy to avoid pre-existing adenovector immunity involves the use of heterologous prime-boost regimens. In this strategy, the priming and boosting are done by using different antigen delivery vehicle and/or vectors derived from either different serotypes of the same species or vectors from completely different host species, for example, priming with DNA and boosting with HAd5 or priming with ChAd68 and boosting with ChAd1 [201]. Studies have shown that the heterologous prime-boost induces more robust immune responses compared to single vaccination or homologous prime-boost immunizations. The cellular immune responses induced by DNA prime and HAd boost were not affected by pre-existing HAd5 immunity. These findings were further confirmed in a clinical trial. Another preclinical study involving *Plasmodium* or SARS antigens encoded by Modified Vaccinia Ankara (MVA)/adenoviral vector as prime/boost showed induction of robust T cell and Ab responses of higher magnitude compared to Ad/DNA regimens. Finally, a ChAd63/ MVA prime-boost strategy is now being evaluated in clinical trials as a vaccine against malaria, HIV, and HCV [191, 202–204]. The heterologous prime/boost strategy seems very effective in circumventing the pre-existing immunity against adenoviral vectors in studies conducted so far; however, the vehicle for priming and/or boosting must be carefully designed and selected.

### *3.3.3. Immunodominance over transgene immunity*

impact of the pre-existing cross-reactive T cells could be far greater in the clinical setting where humans are repeatedly exposed to various serotypes of adenovirus and carry a far broader

Apart from rare human and chimpanzee adenoviruses, several other adenoviruses derived from animals such as bovine, porcine, ovine, canine, and fowl are also being explored for vector development [151, 193–195]. The human population lack nAbs against these adenoviruses, and therefore, the vectors derived from these adenoviruses could be more efficacious in comparison to HAd and ChAd. The mouse models with experimentally induced pre-existing immunity by the administration of HAd vector demonstrated a lack of nAbs

enza virus vaccine demonstrated high efficacy even in the presence of pre-existing HAd5 immunity. There was also no effect of pre-existing HAd5 immunity on transgene expression, immunogenicity, and efficacy in animal models. However, their potential benefits still need

Several studies have reported that different routes of immunizations can negate the detrimental effect of pre-existing immunity. This outcome could be at least partly due to evasion of tissue-resident Ad-specific T cells when using different routes of immunization. Tissueresident CD8 memory T cells remain confined to a specific tissue. As such, they are not systemic and do not prevent Ad vector infection in distant tissues. These tissue-resident T cells are induced by tissue-derived migratory dendritic cells during priming, which activate T cells with specific tissue-homing molecules [196–198]. In a non-human primate model, HAd5 induced protective immune responses by intranasal/intratracheal immunization were not affected by pre-existing HAd5 immunity that had been induced by intramuscular administration of an unrelated HAd5 vector [199]. Although this strategy has shown promising results in animal models, it has not been verified in humans. This strategy is however restricted by limited routes feasible for administration in humans. Moreover, the nAbs induced by adenoviral infection or adenoviral vector-based vaccination can be detected in any part of the body, which may affect the transgene expression and immune responses irrespective of the alternative route used for subsequent adenovector administration. However, nAbs are generally present in blood and are in most cases not cross-reactive to different serotypes or subtypes

Another strategy to avoid pre-existing adenovector immunity involves the use of heterologous prime-boost regimens. In this strategy, the priming and boosting are done by using different antigen delivery vehicle and/or vectors derived from either different serotypes of the same species or vectors from completely different host species, for example, priming with DNA and boosting with HAd5 or priming with ChAd68 and boosting with ChAd1 [201]. Studies have shown that the heterologous prime-boost induces more robust immune

T cells against PAd3 and BAd3 [18]. Furthermore, BAd3- or PAd3-based influ-

diversity and a higher frequency of cross-reactive T cells.

and CD4+

72 Adenoviruses

to be proven in humans.

*3.3.2.2. Routes of immunization*

and Ads from different host species [200].

*3.3.2.3. Heterologous prime-boost strategy*

Adenoviral antigens induce robust antibody and T cell immune responses. Recent studies have shown that adenoviral-derived epitopes can dominate over the transgene-derived epitopes and hinder the induction of transgene-specific immunity. This impairment of transgene-specific immune responses in naive vaccinees is due to immune competition. Epitopes derived from an adenovirus vector were shown to inhibit the induction of HIV GagL85-93-specific CD8<sup>+</sup> T cells [205]. This study demonstrated that competition occurs at the level of responding CD8+ T cells, and co-immunization with an interleukin 2-encoding plasmid restored GagL85-93-specific CD8<sup>+</sup> T cell responses in the presence of an adenoviral hexon486-494 epitope. The IL2, however, could not restore GagL85-93 responsiveness in Ad-based immunization, likely due to the presence of other epitopes in the Ad vector [206]. Another study demonstrated that plasmid DNA, but not adenovirus vector-encoding hepatitis B surface antigen (HBsAg), primed CD8 T cells against subdominant HBsAg epitopes [207]. These studies suggest that adenoviral antigen-specific T-cell immunity is primed efficiently during adenoviral vector-based immunization, which can limit the immunogenicity of adenoviral vector-encoded transgenic antigens. These studies highlight the need for modifications of the vector or the transgene used in immunization to circumvent or dominate over the adenoviral vector-specific epitopes and induce more effective transgenespecific immunity.

### *3.3.4. Heterologous immunity induced by adenoviral antigens*

We discovered an unusual and interesting phenomenon that non-recombinant HAd5 vector induces robust cross-reactive immune responses toward hepatitis C virus (HCV) antigens [91]. Upon further investigation, we found that adenoviral proteins contain extensive homologies with various peptide epitopes derived from HCV antigens. These observations led us to investigate the adenoviral vector-induced HCV cross-reactive immune responses in detail in both mice and humans. In mice, we demonstrated that Ad vector alone can induce potent, broad anti-HCV cross-reactive immunity that can significantly reduce viral load upon challenge with infectious chimeric Vaccinia-HCV. Furthermore, we also detected HCV cross-reactive antibodies and HCV antigen-dependent expression of IFN-γ in T cells from a cohort of HCV-naïve but Ad-immune human individuals. Previous studies have also reported that one pathogen can induce cross-reactive immunity against an unrelated pathogen [91, 208]. This kind of immunity is known as heterologous immunity. Heterologous immunity is a double-edged sword, which can modulate the breadth of the T cell repertoire, influence the memory T cell pool and/or the immune dominance of a specific epitope, and lead to enhanced or diminished immune responses against a pathogen. These observations have significant clinical implications on natural history, immunopathogenesis, and disease outcome in HCV infection. The widespread use of adenoviral vectors in mass vaccination programs might change the immune hierarchy and natural T cell responses against HCV antigens and deviate and/or alter the incidence of HCV infection and immune pathogenesis in an at-risk population. However, a careful evaluation of adenoviral-induced cross-reactive immune responses and their impact on HCV immunity and immunopathology is needed to more accurately ascertain the impact of this phenomenon.

**Conflict of interest**

**Author details**

Shakti Singh1,3, Rakesh Kumar2

Edmonton, Alberta, Canada

**References**

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

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

75

and Babita Agrawal<sup>3</sup>

2 Department of Laboratory Medicine and Pathology, Faculty of Medicine and Dentistry,

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3 Department of Surgery, Faculty of Medicine and Dentistry, University of Alberta,

1 Division of Infectious Disease, Los Angeles Biomedical Research Institute, Harbor-University of California Medical Center, Torrance, California, USA
