*3.2.1. Gene therapy*

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

to deletion of packaging signal. Figure is adapted from Ref. [209].

62 Adenoviruses

**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 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 are ongoing with adenoviral vectors (**Table 1**). The previous generation of adenoviral vectors is still in use for vaccine antigen delivery due to their inherent capability of inducing robust humoral and cellular immune responses.

tolerated upon repeated vaccination and induced robust T-cell responses to HIV antigens. Despite these early findings, the STEP trial had to be terminated prematurely in 2007 due to enhanced acquisition of HIV infection in the vaccine group compared to placebo [137, 138]. A total of 82 cases of HIV infection were recorded in the trial participants, 49 cases were in vaccine recipients and 33 were in placebo group. Another interesting observation was that the HIV infection rate was twofold higher in men with prior adenovirus type 5 infection (Ad5 titers >18) versus placebo recipients [135, 139]. The same HAd5 clade B *gag*/*pol* and *nef* genes-based vaccine was tested in another companion Phambili clinical trial in a South African population. The goal was to investigate whether this vaccine would be efficacious against clade C HIV infections. The participants had different prevalent modes of sexual transmission, different subtypes of HIV-1, and varying Ad5 seroprevalence. Unfortunately, this trial also had to be stopped due to acquisition of HIV infection in 9 females seropositive for HAd5 out of a total of 11 cases. Of these 9 cases, 6 were vaccinees [140, 141]. These results indicated that pre-existing immunity to the Ad5 vector is an important risk factor for HIV acquisition among vaccine recipients. Such profound effects of pre-existing Ad5 immunity on HIV acquisition were not observed in previous studies in non-human primates (NHP) using an adenovirus vector-based vaccine [142]. Several hypotheses were provided for the failure of the STEP trial but none of the hypotheses were proven after experimentation, and the mechanisms for higher HIV acquisition in vaccinees with pre-existing Ad immunity still remain unclear. These results shocked the vaccine community and raised serious questions

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

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65

In 2009, another famous HIV vaccine clinical trial (HVTN phase II) was started [143]. It utilized a heterologous prime-boost strategy, in which vaccinees were first primed with a DNA-based vaccine expressing HIV proteins (envA, envB, envC, gagB, polB, nefB) followed by a booster Ad5 vector vaccine having matching HIV antigens as transgenes. This trial too met with the same fate as STEP, as the vaccine failed to reduce the HIV acquisition rate or attenuate the disease in infected subjects. It was terminated in 2013 prior to completion. However, this study and several other studies provided evidence of superior response rates, induction of broader T cell immune responses with well-accepted tolerance, by heterologous prime-boost vectors compared to homologous vaccination [144]. This started a new vaccination regimen involving priming with one type of adenoviral vector and boosting with another adenoviral vector derived from novel serotypes such as HAd26 and HAd35. This allowed for repeated vaccination and also vaccination in individuals with pre-existing vector immunity [145–149]. Currently, a number of non-human adenoviral vectors such as chimpanzee and bovine are also being utilized to avoid pre-existing vector-specific immu-

Due to the emergence of life-threatening infectious diseases such as Ebola and Zika viruses, an immediate need for vaccines for these pathogens was recognized. These urgent needs attracted researchers toward viral vector platform-based vaccines, especially extensively studied and improved vector technology, and adenoviral vectors became the focus of several vaccines against these infectious diseases [21, 130]. Adenoviral vector-based vaccines are easy to design and to produce on a mass scale, which is of paramount significance for clinical

regarding the fate of adenoviral vectors in vaccine approaches.

nity [18, 21, 150–152].

### *3.2.2. Vaccine vector*

As described earlier, adenoviruses activate several innate immune signaling pathways that result in the secretion of a number of proinflammatory cytokines. These proinflammatory cytokines pave the way for effective immune cell stimulation and result in the induction of robust adaptive humoral and cellular immune responses. To resolve infections with intracellular pathogens such as viruses, CD8+ cytotoxic T lymphocyte (CTL) responses are critical. Transgene antigens carried by adenoviral vectors are presented to T cells via MHC class I molecules, and therefore, they induce efficient and robust CTL responses. The CTLs efficiently recognize and kill virus-infected cells, intracellular pathogens, and cancerous cells. These properties make adenoviral vectors promising as vaccine vectors. A number of human clinical trials have been conducted for adenoviral vector-based vaccines against different infectious diseases including Ebola virus, Zika virus, influenza viruses, HIV, *Mycobacterium tuberculosis*, and malaria [21, 130].

An HAd5 vector-based HIV vaccine containing clade B sequences of *gag*/*pol* and *nef* genes was tested in several clinical trials by Merck during 2003–2006 [131]. These studies demonstrated that a majority (80%) of vaccine recipients induced T cells with a magnitude of 275–300 IFN-γ producing cells per million peripheral blood mononuclear cells (PBMCs), where about 50% vaccinees had detectable and durable HIV-specific CD8<sup>+</sup> T and CD4<sup>+</sup> T cells. This is far greater than any other T cell vaccine at that time [132–135]. These results were very encouraging and led to a multinational STEP trial involving about 3000 subjects in 2005 [136, 137]. The early results of this vaccine indicated that vaccine was well


**Table 1.** Gene therapy: adenoviral vectors in clinical trial.

tolerated upon repeated vaccination and induced robust T-cell responses to HIV antigens. Despite these early findings, the STEP trial had to be terminated prematurely in 2007 due to enhanced acquisition of HIV infection in the vaccine group compared to placebo [137, 138]. A total of 82 cases of HIV infection were recorded in the trial participants, 49 cases were in vaccine recipients and 33 were in placebo group. Another interesting observation was that the HIV infection rate was twofold higher in men with prior adenovirus type 5 infection (Ad5 titers >18) versus placebo recipients [135, 139]. The same HAd5 clade B *gag*/*pol* and *nef* genes-based vaccine was tested in another companion Phambili clinical trial in a South African population. The goal was to investigate whether this vaccine would be efficacious against clade C HIV infections. The participants had different prevalent modes of sexual transmission, different subtypes of HIV-1, and varying Ad5 seroprevalence. Unfortunately, this trial also had to be stopped due to acquisition of HIV infection in 9 females seropositive for HAd5 out of a total of 11 cases. Of these 9 cases, 6 were vaccinees [140, 141]. These results indicated that pre-existing immunity to the Ad5 vector is an important risk factor for HIV acquisition among vaccine recipients. Such profound effects of pre-existing Ad5 immunity on HIV acquisition were not observed in previous studies in non-human primates (NHP) using an adenovirus vector-based vaccine [142]. Several hypotheses were provided for the failure of the STEP trial but none of the hypotheses were proven after experimentation, and the mechanisms for higher HIV acquisition in vaccinees with pre-existing Ad immunity still remain unclear. These results shocked the vaccine community and raised serious questions regarding the fate of adenoviral vectors in vaccine approaches.

are ongoing with adenoviral vectors (**Table 1**). The previous generation of adenoviral vectors is still in use for vaccine antigen delivery due to their inherent capability of inducing

As described earlier, adenoviruses activate several innate immune signaling pathways that result in the secretion of a number of proinflammatory cytokines. These proinflammatory cytokines pave the way for effective immune cell stimulation and result in the induction of robust adaptive humoral and cellular immune responses. To resolve infections with intracel-

Transgene antigens carried by adenoviral vectors are presented to T cells via MHC class I molecules, and therefore, they induce efficient and robust CTL responses. The CTLs efficiently recognize and kill virus-infected cells, intracellular pathogens, and cancerous cells. These properties make adenoviral vectors promising as vaccine vectors. A number of human clinical trials have been conducted for adenoviral vector-based vaccines against different infectious diseases including Ebola virus, Zika virus, influenza viruses, HIV, *Mycobacterium* 

An HAd5 vector-based HIV vaccine containing clade B sequences of *gag*/*pol* and *nef* genes was tested in several clinical trials by Merck during 2003–2006 [131]. These studies demonstrated that a majority (80%) of vaccine recipients induced T cells with a magnitude of 275–300 IFN-γ producing cells per million peripheral blood mononuclear cells (PBMCs),

cells. This is far greater than any other T cell vaccine at that time [132–135]. These results were very encouraging and led to a multinational STEP trial involving about 3000 subjects in 2005 [136, 137]. The early results of this vaccine indicated that vaccine was well

> transmembrane conductance regulator (CFTR) gene

Pigment epithelium-derived factor (PEDF) protein

factor D (VEGF-D) gene

**Modification Transgene Target/**

where about 50% vaccinees had detectable and durable HIV-specific CD8<sup>+</sup>

(hAQP1)

factor B (PDGF-B)

cytotoxic T lymphocyte (CTL) responses are critical.

**condition**

Macular degeneration

Parotid salivary dysfunction

Angina pectoris/ myocardial infarction

T and CD4<sup>+</sup>

**Phase ClinicalTrials identifier**

I NCT00372320

I NCT00109499

I NCT01002430

Cystic fibrosis I NCT00004779

Varicose ulcer I NCT00000431

T

robust humoral and cellular immune responses.

lular pathogens such as viruses, CD8+

*tuberculosis*, and malaria [21, 130].

**Adenoviral vector (biologic)**

4 HAd5-PEDF

(AdGVPEDF.11D)

1 HAd5-CB-CFTR E1 deleted Cystic fibrosis

**Table 1.** Gene therapy: adenoviral vectors in clinical trial.

2 HAd5-hAQP1 E1 deleted Human aquaporin-1

3 HAd5-PDGF-B E1 deleted Platelet-derived growth

E1, E3 and E4 deleted

5 HAd5-VEGF E1–E3-deleted Vascular endothelial growth

**S. no.**

*3.2.2. Vaccine vector*

64 Adenoviruses

In 2009, another famous HIV vaccine clinical trial (HVTN phase II) was started [143]. It utilized a heterologous prime-boost strategy, in which vaccinees were first primed with a DNA-based vaccine expressing HIV proteins (envA, envB, envC, gagB, polB, nefB) followed by a booster Ad5 vector vaccine having matching HIV antigens as transgenes. This trial too met with the same fate as STEP, as the vaccine failed to reduce the HIV acquisition rate or attenuate the disease in infected subjects. It was terminated in 2013 prior to completion. However, this study and several other studies provided evidence of superior response rates, induction of broader T cell immune responses with well-accepted tolerance, by heterologous prime-boost vectors compared to homologous vaccination [144]. This started a new vaccination regimen involving priming with one type of adenoviral vector and boosting with another adenoviral vector derived from novel serotypes such as HAd26 and HAd35. This allowed for repeated vaccination and also vaccination in individuals with pre-existing vector immunity [145–149]. Currently, a number of non-human adenoviral vectors such as chimpanzee and bovine are also being utilized to avoid pre-existing vector-specific immunity [18, 21, 150–152].

Due to the emergence of life-threatening infectious diseases such as Ebola and Zika viruses, an immediate need for vaccines for these pathogens was recognized. These urgent needs attracted researchers toward viral vector platform-based vaccines, especially extensively studied and improved vector technology, and adenoviral vectors became the focus of several vaccines against these infectious diseases [21, 130]. Adenoviral vector-based vaccines are easy to design and to produce on a mass scale, which is of paramount significance for clinical use. Therefore, three adenoviral vector-based vaccines encoding Ebola virus glycoprotein, ChAd3-ZEBOV1 from GlaxoSmithKline [153], Ad26-ZEBOV/MVA-BN-Filo2 from Johnson & Johnson [154], and HAd5 from the Chinese federal agency [155] were quickly generated and tested in macaques. Each of them proved to be well tolerated, immunogenic, and protective in macaques. All these vaccines were also well tolerated, safe, and immunogenic in phase I clinical trials, and the ChAd3- and ChAd26/MVA-based vaccines progressed further into phase II and phase III efficacy trials [156, 157]. The Chinese Ad5-based Ebola vaccine showed less efficacy in phase I clinical trial in individuals with pre-existing adenoviral immunity [158]. Beside these, a chimpanzee adenoviral vector ChAd63 prime/MVA boost-based malaria vaccine, which contains *Plasmodium falciparum*-derived ME-TRAP antigen, showed a significant enhancement in antigen-specific T cell responses and partial protection against malarial parasites in a phase I clinical trial [156, 159].

**S. no.** **Adenoviral vector (biologic)**

MVA-EBOV-Z-BN-Filo

3 ChAd3-EBO-Z/

Prime/Boost

4 Ad26-EBOV-Z/MVA-BN-Filo Prime/boost

5 Ad26-EBOV-Z/MVA-BN-Filo Prime/boost

6 HAd6-Nsmut/ VhAd3NSmut or CHAd3NSmut/ HAd6NSmut Prime/

boost

7 AdCh3NSmut/ Ad6NSmut

10 HAd4-HA5-Vtn HA E3-partial

(NMRC-M3V-Ad-PfCA)

11 HAd35-CSP/HAd26- CSP Vectors Prime/

13 ChAd63-ME-TRAP/ MVA-ME-TRAP poxvirus Prime/boost

14 HAd35-TB Antigens/ MVA85A Prime/boost (AERAS-402, BCG)

15 HAd35-TB Antigens (AERAS-402)

boost

12 HAd5

**Modification Transgene Target/**

E1-deleted Glycoprotein/envelope filovirus

1 HAd5-EBOV E1-deleted Glycoprotein Ebola virus

2 ChAd3-EBO-Z E1-deleted Glycoprotein Ebola virus

E1 and E3 deleted

E1 and E3 deleted

8 HAd5-HA (VXA-1.1) E1-deleted Hemagglutinin and

deletion

E1, E4 deleted, E3 partially deleted

E1-deleted, HAd5 E4 orf6 replaced

E1-deleted, HAd5 E4 orf6 replaced

E1 and E3 deleted

9 HAd5-HA E1/E3-deleted Hemagglutinin Influenza

**condition**

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

disease

disease

disease

disease

Influenza H1N1

H5N1

H5N1

Glycoprotein Ebola virus

Glycoprotein Ebola virus

E1-deleted Non-structural protein Hepatitis C I NCT01070407

E1-deleted Non-structural protein Hepatitis C I NCT01094873

double-stranded RNA as an adjuvant

Circumsporozoite (CSP) antigen

Circumsporozoite (CSP) antigen, apical membrane antigen 1

(pre-erythrocytic thrombospondinrelated adhesion protein)

TB antigens: Ag85A, Ag85B, and TB10.4

TB antigens: Ag85A, Ag85B, and TB10.4

(AMA1)

E1-deleted ME-TRAP antigen

Hemagglutinin Influenza

Ebola virus disease

**Phase ClinicalTrials identifier**

67

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

II NCT02575456

I/II NCT02289027

I NCT02267109

I NCT02891980

III NCT02661464

II NCT02918006

I NCT00755703

I NCT01006798

Malaria I/II NCT01397227

Malaria I/II NCT00392015

Malaria I NCT01373879

Tuberculosis I NCT01683773

Tuberculosis II NCT02414828

Despite initial setbacks, adenoviral vector-based vaccines are still very attractive and promising vaccine platforms. Currently, several adenoviral vector-based vaccines are in different stages of clinical development (**Table 2**).

### *3.2.3. Cancer immunotherapy*

Several DNA viruses such as reo, measles, herpes simplex, Newcastle disease, and vaccinia have been tested in clinical trials for anticancer immunotherapy. The mechanism of anticancer activity of these viruses is multipronged. One mechanism involves selective infection and replication in tumor cells where expression of viral antigens or oncogenes inside the cancer cell changes the tumor microenvironment by inducing proinflammatory cytokines. These subsequently attract immune cells to the tumors eventually resulting in lysis of the tumor cells. Another mechanism uses vectors to deliver gene(s) whose expression results in the apoptosis of cancer cells or lysis of cancer cells due to replication by replicationcompetent viral vectors [160, 161]. Adenoviral vector technologies have progressed to the clinical stage for various cancers and also have been approved in some countries for use in human [162–164].

Various anticancer approaches have been tested using adenoviral vectors. One of these approaches depends on the induction of immune responses by delivering specific tumorassociated antigen as a vaccine, which activates immune cells against the tumor [165–168]. Due to immunogenic properties of adenoviral proteins such as capsid, adenoviral vectors induce robust CTL responses, which eventually kill the tumor cells expressing these tumor antigens. However, this vaccination strategy has shown limited success in cancer.

Another approach uses conditional replicative adenoviral vector (CRAd) to preferentially replicate inside a tumor cell and eventually lyse it through a lytic replication [162]. This strategy takes advantage of the conducive nature of cancer cells toward adenoviruses. Adenoviral vectors have been modified to efficiently carry out oncolytic replication in cancer cells while limiting their replication in healthy cells [9, 169]. An adenoviral vector with a partial E1B gene deficiency called ONYX-015 became the first ever adenoviral vector to enter clinical trial in 1996 [169]. The ONYX-015 is unable to replicate in healthy cells expressing p53 but replicates


use. Therefore, three adenoviral vector-based vaccines encoding Ebola virus glycoprotein, ChAd3-ZEBOV1 from GlaxoSmithKline [153], Ad26-ZEBOV/MVA-BN-Filo2 from Johnson & Johnson [154], and HAd5 from the Chinese federal agency [155] were quickly generated and tested in macaques. Each of them proved to be well tolerated, immunogenic, and protective in macaques. All these vaccines were also well tolerated, safe, and immunogenic in phase I clinical trials, and the ChAd3- and ChAd26/MVA-based vaccines progressed further into phase II and phase III efficacy trials [156, 157]. The Chinese Ad5-based Ebola vaccine showed less efficacy in phase I clinical trial in individuals with pre-existing adenoviral immunity [158]. Beside these, a chimpanzee adenoviral vector ChAd63 prime/MVA boost-based malaria vaccine, which contains *Plasmodium falciparum*-derived ME-TRAP antigen, showed a significant enhancement in antigen-specific T cell responses and partial protection against malarial para-

Despite initial setbacks, adenoviral vector-based vaccines are still very attractive and promising vaccine platforms. Currently, several adenoviral vector-based vaccines are in different

Several DNA viruses such as reo, measles, herpes simplex, Newcastle disease, and vaccinia have been tested in clinical trials for anticancer immunotherapy. The mechanism of anticancer activity of these viruses is multipronged. One mechanism involves selective infection and replication in tumor cells where expression of viral antigens or oncogenes inside the cancer cell changes the tumor microenvironment by inducing proinflammatory cytokines. These subsequently attract immune cells to the tumors eventually resulting in lysis of the tumor cells. Another mechanism uses vectors to deliver gene(s) whose expression results in the apoptosis of cancer cells or lysis of cancer cells due to replication by replicationcompetent viral vectors [160, 161]. Adenoviral vector technologies have progressed to the clinical stage for various cancers and also have been approved in some countries for use in

Various anticancer approaches have been tested using adenoviral vectors. One of these approaches depends on the induction of immune responses by delivering specific tumorassociated antigen as a vaccine, which activates immune cells against the tumor [165–168]. Due to immunogenic properties of adenoviral proteins such as capsid, adenoviral vectors induce robust CTL responses, which eventually kill the tumor cells expressing these tumor

Another approach uses conditional replicative adenoviral vector (CRAd) to preferentially replicate inside a tumor cell and eventually lyse it through a lytic replication [162]. This strategy takes advantage of the conducive nature of cancer cells toward adenoviruses. Adenoviral vectors have been modified to efficiently carry out oncolytic replication in cancer cells while limiting their replication in healthy cells [9, 169]. An adenoviral vector with a partial E1B gene deficiency called ONYX-015 became the first ever adenoviral vector to enter clinical trial in 1996 [169]. The ONYX-015 is unable to replicate in healthy cells expressing p53 but replicates

antigens. However, this vaccination strategy has shown limited success in cancer.

sites in a phase I clinical trial [156, 159].

stages of clinical development (**Table 2**).

*3.2.3. Cancer immunotherapy*

66 Adenoviruses

human [162–164].


more effective when administered in combination with standard chemotherapy [169, 170]. A similar adenoviral vector named Oncorine or H101, developed by Shanghai Sunway Biotech, was approved by the Chinese Food and Drug Administration agency for the treatment of head and neck cancer [171, 172]. To further enhance the efficacy, potency, and specificity of the oncolytic adenoviral vectors, a new generation of adenoviral vectors is being tested. These new adenoviral vector systems carry a suicide gene like HSV thymidine kinase or a cytotoxic prodrug under the control of tumor gene/antigen promotor like prostate antigen promotor

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

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69

In some studies, replication-deficient or replication-competent adenoviral vectors were used to deliver transgenes, which express a tumor suppressor protein or cytotoxic/suicide protein that induces cell cycle arrest or a death cascade [176, 177]. More than 50% of cancers have a mutation in tumor suppressor gene p53. Advexin is a replication-deficient adenoviral vector that expresses p53 through a CMV promotor. It was tested in both preclinical and more than a dozen phase I/II clinical trials and proved to be well tolerated and efficacious against colorectal cancer, hepatocellular carcinoma (HCC), non-small cell lung cancer (NSCLC), prostate cancer, breast cancer, ovarian cancer, bladder cancer, glioma, and squamous cell carcinoma of the head and neck [171, 178, 179]. Gendicine, a similar adenovirus vector developed by a Chinese Biotech Company, Shenzhen SiBiono GeneTech, differs only in that its transgene promotor is from Rous Sarcoma Virus. In 2003, Gendicine was approved by the Chinese Food and Drug Administration agency as a first-ever gene therapy product to be used in combination with chemotherapy to treat head and neck squamous cell carcinoma [4, 178, 180]. Since then, Gendicine has been tested in a number of clinical trials against different types of cancers such as HCC, NSCLC, malignant glioma, and epithelial ovarian carcinoma. It is reported to be well tolerated and provide progression-free long-term survival benefits in combination regimens when compared to standard therapies alone [4, 178, 180, 181]. Therefore, adenoviral vectors have been clinically successful in anticancer therapy and have shown tremendous potential in the treatment of several cancer types [4, 171, 180, 181]. However, there is still scope for further

Development of adenoviral vectors has come a long way since their first use. Currently, different types of adenoviral vectors are available for different applications. In the beginning, a high prevalence of pre-existing immunity to adenoviral vectors was considered as a serious concern for their use in mass vaccination and gene therapeutic applications. Further, immunogenicity, cellular toxicity, and oncogenesis were also major obstacles in gene therapy applications. Many other concerns, such as the possibility of vectors regaining replication competence, non-specificity, immunodominance of adenoviral antigens over the vaccine transgene antigen(s), immune modulation by viral antigens, heterologous immunity with other pathogens, are still evident in many adenoviral vector-based vaccine and gene therapy

improvements in clinical efficacy and safety (**Table 3**).

**3.3. Challenges and solutions to adenoviral vectors use**

approaches [182]. These are discussed in the following sections.

[173–175].

**Table 2.** Vaccine delivery: adenoviral vectors in clinical trials.

in p53-deficient tumor cells and results in the lysis of the cell, taking advantage of the cancer cell environment that supports vector replication [9]. The ONYX-015 has been proven to be safe and well tolerated in patients with various advanced cancers and is reported to be even more effective when administered in combination with standard chemotherapy [169, 170]. A similar adenoviral vector named Oncorine or H101, developed by Shanghai Sunway Biotech, was approved by the Chinese Food and Drug Administration agency for the treatment of head and neck cancer [171, 172]. To further enhance the efficacy, potency, and specificity of the oncolytic adenoviral vectors, a new generation of adenoviral vectors is being tested. These new adenoviral vector systems carry a suicide gene like HSV thymidine kinase or a cytotoxic prodrug under the control of tumor gene/antigen promotor like prostate antigen promotor [173–175].

In some studies, replication-deficient or replication-competent adenoviral vectors were used to deliver transgenes, which express a tumor suppressor protein or cytotoxic/suicide protein that induces cell cycle arrest or a death cascade [176, 177]. More than 50% of cancers have a mutation in tumor suppressor gene p53. Advexin is a replication-deficient adenoviral vector that expresses p53 through a CMV promotor. It was tested in both preclinical and more than a dozen phase I/II clinical trials and proved to be well tolerated and efficacious against colorectal cancer, hepatocellular carcinoma (HCC), non-small cell lung cancer (NSCLC), prostate cancer, breast cancer, ovarian cancer, bladder cancer, glioma, and squamous cell carcinoma of the head and neck [171, 178, 179]. Gendicine, a similar adenovirus vector developed by a Chinese Biotech Company, Shenzhen SiBiono GeneTech, differs only in that its transgene promotor is from Rous Sarcoma Virus. In 2003, Gendicine was approved by the Chinese Food and Drug Administration agency as a first-ever gene therapy product to be used in combination with chemotherapy to treat head and neck squamous cell carcinoma [4, 178, 180]. Since then, Gendicine has been tested in a number of clinical trials against different types of cancers such as HCC, NSCLC, malignant glioma, and epithelial ovarian carcinoma. It is reported to be well tolerated and provide progression-free long-term survival benefits in combination regimens when compared to standard therapies alone [4, 178, 180, 181]. Therefore, adenoviral vectors have been clinically successful in anticancer therapy and have shown tremendous potential in the treatment of several cancer types [4, 171, 180, 181]. However, there is still scope for further improvements in clinical efficacy and safety (**Table 3**).

### **3.3. Challenges and solutions to adenoviral vectors use**

in p53-deficient tumor cells and results in the lysis of the cell, taking advantage of the cancer cell environment that supports vector replication [9]. The ONYX-015 has been proven to be safe and well tolerated in patients with various advanced cancers and is reported to be even

Env gp140 HIV

HIV-1 Mos1Env HIV

Hiv gag, pol and Env HIV

mosaic HIV Gag antigen, HIV clade C Env protein (gp150

1086.C)

**Modification Transgene Target/**

HIV antigens gp140(A), gp140(B) dv12, gp140(C) and GagPol(B)

HIV antigens gp140(A), gp140(B) dv12, gp140(C) and GagPol(B)

HIV antigens gp140(A), gp140(B) dv12, gp140(C) and GagPol(B)

E1-deleted Gag, pol and Nef antigens

E1, E4 and partial E3 deleted

E1, E4 and partial E3 deleted

E1, E4 and partial E3 deleted

E1 and E3 deleted

Replication competent

E3 or E3/E4 deleted

E3 or E3/E4 deleted

**Table 2.** Vaccine delivery: adenoviral vectors in clinical trials.

**condition**

HIV infections

HIV infections

HIV infections

HIV infections

infections

infections

infections

HIV infections **Phase ClinicalTrials identifier**

I NCT01549509, NCT00119873, NCT00091416, NCT00709605, NCT00102089

I/II NCT00123968, NCT00125970

II NCT00865566

I NCT00801697

I NCT00618605, NCT01103687

I NCT02771730, NCT01989533

I NCT02366013

I/II NCT02919306

**S. no.**

68 Adenoviruses

**Adenoviral vector (biologic)**

expressing Gag, Plo and Nef +Four HAd5 vectors for four HIVAntigens (VRC-HIVDNA016-00-VP/ VRC-HIVADV014- 00-VP), DNA + HAd5/ HAd5 Prime/boost

18 Plasmid DNA vaccine/ HAd5-HIV-1 (VRC-HIVDNA016-00-VP/ VRC-HIVADV014- 00-VP) prime/boost

> HAd35/HAd5 (VRC-HIVDNA044-00-VP/ VRC-HIVADV027- 00-VP/VRC-HIVADV038-00-VP) HAd35/HAd5 prime/ boost or DNA/HAd5 prime/boost or DNA/ HAd35 prime/boost

19 DNA Vaccine/

20 HAd26 (HAd26. ENVA.01)

21 HAd4-mgag, HAd4- EnvC150 alone or combination

22 rcAd26.MOS1.HIV-Env

23 Ad26.Mos.HIV (Ad26. Mos.1.Env + Ad26. Mos1.Gag-Pol + Ad26. Mos2.Gag-Pol)/MVA-Mosaic prime/boost

1

16 Four HAd5 vectors for four HIVAntigens (VRC-HIVADV014- 00-VP/VRC-HIVADV014-00-VP)

17 Plasmid DNA

Development of adenoviral vectors has come a long way since their first use. Currently, different types of adenoviral vectors are available for different applications. In the beginning, a high prevalence of pre-existing immunity to adenoviral vectors was considered as a serious concern for their use in mass vaccination and gene therapeutic applications. Further, immunogenicity, cellular toxicity, and oncogenesis were also major obstacles in gene therapy applications. Many other concerns, such as the possibility of vectors regaining replication competence, non-specificity, immunodominance of adenoviral antigens over the vaccine transgene antigen(s), immune modulation by viral antigens, heterologous immunity with other pathogens, are still evident in many adenoviral vector-based vaccine and gene therapy approaches [182]. These are discussed in the following sections.


common human adenoviruses (HAd5 and HAd2) bind to coxsackie adenovirus receptor (CAR) present on many different cell types including epithelial, endothelial, hepatocytes, myoblasts, and heart muscle cells. Some cells such as lymphocytes do not express CAR themselves but harbor CAR-recognizing adenoviruses. Adenovirus from subgroup B such as HAd35 do not bind to CAR but recognize another complement regulatory receptor CD46 present on most nucleated human cells, hematopoietic stem cells, and dendritic cells. Another subgroup B adenovirus HAd3 binds to CD80 and CD86 costimulatory molecules on antigen-presenting cells [8, 166, 183, 184]. Other cellular receptors such as integrin αvβ5, heparin sulfate proteoglycans, and sialic acid have also been reported to aid adenoviral

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

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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

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 immu-

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

adenoviral vectors, several strategies are being employed as discussed below.

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

nogenicity and efficacy of the HAd35 vectors [188, 189].

entry to the cells [30–32, 185].

*3.3.2. Pre-existing adenoviral immunity*

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

### *3.3.1. Tissue tropism and transgene expression*

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 common human adenoviruses (HAd5 and HAd2) bind to coxsackie adenovirus receptor (CAR) present on many different cell types including epithelial, endothelial, hepatocytes, myoblasts, and heart muscle cells. Some cells such as lymphocytes do not express CAR themselves but harbor CAR-recognizing adenoviruses. Adenovirus from subgroup B such as HAd35 do not bind to CAR but recognize another complement regulatory receptor CD46 present on most nucleated human cells, hematopoietic stem cells, and dendritic cells. Another subgroup B adenovirus HAd3 binds to CD80 and CD86 costimulatory molecules on antigen-presenting cells [8, 166, 183, 184]. Other cellular receptors such as integrin αvβ5, heparin sulfate proteoglycans, and sialic acid have also been reported to aid adenoviral entry to the cells [30–32, 185].
