key: cord-0838807-4zrs8bhx
authors: Sakurai, Fuminori; Tachibana, Masashi; Mizuguchi, Hiroyuki
title: Adenovirus vector-based vaccine for infectious diseases
date: 2021-11-12
journal: Drug Metab Pharmacokinet
DOI: 10.1016/j.dmpk.2021.100432
sha: d12fe3302523419aa49e726836968e0e0c315be6
doc_id: 838807
cord_uid: 4zrs8bhx

Replication-incompetent adenovirus (Ad) vectors have been widely used as gene delivery vehicles in both gene therapy studies and basic studies for gene function analysis due to their highly advantageous properties, which include high transduction efficiencies, relatively large capacities for transgenes, and high titer production. In addition, Ad vectors induce moderate levels of innate immunity and have relatively high thermostability, making them very attractive as potential vaccine vectors. Accordingly, it is anticipated that Ad vectors will be used in vaccines for the prevention of infectious diseases, including Ebola virus disease and acquired immune deficiency syndrome (AIDS). Much attention is currently focused on the potential use of an Ad vector vaccine for coronavirus disease 2019 (COVID-19). In this review, we describe the basic properties of an Ad vector, Ad vector-induced innate immunity and immune responses to Ad vector-produced transgene products. Development of novel Ad vectors which can overcome the drawbacks of conventional Ad vector vaccines and clinical application of Ad vector vaccines to several infectious diseases are also discussed.

Coronavirus disease 2019 (COVID-19) and the severe acute respiratory coronavirus 2 (SARS-CoV-2) which causes it have reminded us that infectious diseases are still a major global threat to human beings.

Vaccines are a powerful tool to fight various types of pathogens and to prevent infection and progression of infectious diseases. Conventional vaccines are commonly composed of inactivated or attenuated pathogens, and proteins derived from pathogens. Conventional vaccines mimic the infection with pathogens, resulting in the induction of immune responses to pathogen-derived proteins. Although conventional vaccines have been successfully used to induce durable protective immunity against many infectious diseases, conventional vaccine development is not a suitable approach to counter the outbreak of emerging infectious diseases caused by largely unknown pathogens. The development of conventional vaccines takes a long time, usually more than 10 years. To counter the outbreak of an emerging infectious disease, efficient and rapid production of vaccines is indispensable. In addition, mutant strains of pathogens which can escape from the effects of vaccines are often generated. Live attenuated vaccines possess a potential risk for reversion to virulence. Inactivated viral vaccines do not always induce protective effects. Cultivation and propagation of pathogens are indispensable for the production of live attenuated or inactivated vaccines, but these processes are often associated with numerous difficulties, including the requirements of facilities with a high biosafety level and large scale production. In order to circumvent these hurdles, novel approaches or platforms for vaccines are highly anticipated.

Novel types of vaccines composed of DNA or mRNA encoding pathogen proteins are expected to be J o u r n a l P r e -p r o o f where it forms a tight junction, it is difficult to access to CAR following intranasal or intratracheal administration of an Ad vector [22] . CD46 is also a basolateral protein [23] . Further investigation into the involvement of infection receptors in Ad vector-mediated vaccination could provide important clues for the improvement of Ad vector vaccines.

Activation of innate immunity is highly important for activation of adaptive immunity. Innate immunityinduced production of inflammatory cytokines and interferons (IFNs) leads to activation of immune cells, including T cells and B cells, followed by activation of adaptive immunity. Conventional vaccines often contain adjuvants which can efficiently activate innate immunity via stimulation of pattern recognition receptors (PPRs). An Ad vector moderately activates innate immunity without adjuvants, because the components of an Ad particle are recognized by various types of PRRs, including toll-like receptors (TLRs), retinoic acid-inducible gene-I (RIG-I)-like receptors, and cyclic guanine adenine synthase (cGAS) [24] , and serve as an adjuvant. On the other hand, an Ad vector does not induce severe innate immune responses, such as cytokine storm or severe damages in the transduced cells. Ad vector-mediated innate immunity activation levels are appropriate to activate adaptive immunity to transgene products without severe side effects. The following PRR families have been demonstrated to be involved in Ad vector-induced innate immunity.

Toll-like receptors. TLRs are a major PRR family consisting of 10 toll-like receptor (TLR) members in humans and 12 members in mice. Each TLR family member recognizes different ligands. Among the TLR family J o u r n a l P r e -p r o o f members, TLR9, which is mainly located on the endosomal membrane and recognizes unmethylated CpGmotif-containing DNA, recognizes the Ad vector genome in the endosomes following internalization of Ad vector particles into cells, leading to activation of the NF-kB signal and IFN regulatory factor (IRF) signal [25] [26] [27] . TLR9 is mainly expressed on dendritic cells (DCs), NK cells, and macrophages. In addition to TLR9, TLR2 and TLR4 have also been shown to be involved in Ad vector-induced innate immunity [28, 29] . TLR2 is mainly expressed on DCs, monocytes, and T cells and recognizes molecules with diacyl and triacylglycerol moieties, proteins and polysaccharides. Ad vector-mediated inductions of transgene product-specific IgM, IgG2, IgG3, and IgA were down-regulated in TLR2-knockout (KO) mice [28] , although it is unclear which Ad vector components were recognized by TLR2. TLR4 is mainly expressed on DCs, monocytes, and T cells, and recognizes various damage-associated molecular patterns (DAMPs). Lactoferrin, which is a ligand of TLR4, binds to the capsids of Ad vectors, leading to activation of innate immunity via TLR4 [30] . Transgene productspecific IgG3 levels were lower in TLR4-KO mice than wild-type mice after intravenous injection of Ad vectors [29] . All TLR family members except for TLR3 require MyD88, which is a crucial adaptor molecule for TLR signal transduction. Both transgene product-specific antibody production and cytotoxix T lymphocyte (CTL) induction were attenuated in MyD88-KO mice following intramuscular vaccination with an Ad vector vaccine [31] , indicating that TLR-MyD88 signaling is highly important for Ad vector-mediated vaccination.

cGAS. cGAS is a PRR located in the cytosol [32] . The Ad vector genome binds to and activates cGAS in the cytosol following endosomal escape into the cytosol [33] . cGAS is ubiquitously expressed in a variety of cells.

Binding of Ad vector genome to cGAS results in activation of the NF-kB signal and IRF signal. Ad vector-J o u r n a l P r e -p r o o f induced inflammatory cytokine production and expression levels of activation markers on DCs were largely reduced in cGAS-KO mice, but anti-Ad antibody production levels were comparable between wild-type and cGAS-KO mice following Ad vector vaccination [33] .

RIG-I-like receptors. RIG-I-like receptors, including RIG-I and melanoma differentiation association gene-5 (MDA5), are also located in the cytosol [34] . RIG-I and MDA5, which are expressed in a wide variety of cells, mainly recognize pathogen-derived double-stranded RNAs, leading to activation of NF-KB and IRF signals.

Virus-associated RNAs (VA-RNAs) were shown to be recognized by RIG-I-like receptors [35, 36] . VA-RNAs are approximately 160-nt long non-coding RNAs transcribed from not only the wild-type Ad genome but also the replication-incompetent Ad vector genome [35] . Ad vector-mediated activation of innate immunity in primary mouse cells genetically lacking mitochondrial antiviral-signaling (MAVS) (also called IFN- promoter stimulator 1 (IPS-1)), which is a signal adaptor protein downstream of RIG-I and MDA5, was significantly lower than that in wild-type cells [35] . CTL induction was attenuated in the mucosal compartment of MAVS-KO mice following Ad vector vaccination [37] , although transgene product-specific antibody production levels were comparable between wild-type and MAVS-KO mice following immunization with a modified Ad vector displaying the antigen epitopes [38] . These results suggested that a RIG-I-like receptor pathway is crucial for Ad vector vaccine-mediated CTL induction in the mucosal compartment.

In addition to the PRRs described above, the other PRRs, including absent in melanoma 2 (AIM2) and NALP3, have been reported to be involved in Ad vector-induced innate immunity [39, 40]. As described above, the involvement of PRRs in the effects of Ad vector-mediated vaccines are highly complex and highly J o u r n a l P r e -p r o o f dependent on the administration routes, types of antigens, injected doses, and types of Ad vectors (most of the studies described above were performed by using an Ad5 vector). Further examination is necessary to fully understand the involvement of PRRs in Ad vector-mediated vaccination.

Ad vectors can induce transgene product-specific immune responses-that is, induction of not only antibody production, but also CTLs. Ad vector-expressing transgene products are recognized as non-self and are eliminated by the immune system. Following administration of Ad vectors, transgenes are expressed in non-immune cells (e.g., muscle cells, fibroblasts) and/or immune cells (e.g., DCs, macrophages). When Ad vector vaccines mediate transgene expression in non-immune cells, transgene products are released from the cells, followed by uptake of transgene products by antigen-presenting cells (APCs), leading to mainly production of transgene product-specific antibodies. Ad vector vaccines elicit various isotypes and subclasses of transgene product-specific antibodies following administration. In addition, intramuscular administration of Ad vector vaccines leads to induction of not only systemic humoral immunity but also certain levels of mucosal humoral immunity, although mucosal administration of an Ad vector vaccine has been shown to induce mucosal immunity more efficiently than intramuscular administration [41-43].

When the transgenes are expressed in immune cells, antigen presentation occurs in immune cells, leading to mainly CTL induction. Transgene expression in monocytes/macrophages and several DC subsets, such as CD8 + DCs, langerin + dermal DCs (dDCs), plasmacytoid DCs (pDCs), and inflammatory DCs (inf J o u r n a l P r e -p r o o f DCs) was found following intravenous, intramuscular, and intranasal administration [44] [45] [46] . Induction of CTLs is highly important to eliminate pathogen-infected cells. Moreover, an Ad vector vaccine induced CD8 + T cells showing polyfunctional phenotypes following intramuscular administration [47, 48] . Ad vector vaccines induce strong CTL responses in not only the systemic compartment in but also the mucosal compartment [37, 49, 50] . This is an attractive advantage of an Ad vector vaccines, because pathogens often infect and invade from the mucosal compartment. Hemmi et al. suggested the following mechanism of Ad vector vaccine-mediated mucosal CTL induction following intramuscular administration as follows [49] .

First, inflammatory monocytes are recruited to the muscle after intramuscular administration of Ad vectors.

Then, the monocytes take up the transgene products, leading to differentiation to inf DCs. inf DCs migrate to the draining LNs (dLNs), followed by induction of T helper 17 (Th17) cells. Th17 cells migrate to the gutmucosa, resulting in the proliferation of antigen-specific CTLs in the mucosal compartment. In addition, a chimpanzee Ad-based vector, ChAdOx1, which is used as a platform of SARS-CoV-2 vaccine, has been shown to efficiently activate mucosal-associated invariant T (MAIT) cells, which are the innate lymphoid cells in the J o u r n a l P r e -p r o o f mucosal compartment and play an important role in mucosal immunity, via the following mechanism [46] .

First, ChAdOx1 infects pDCs, leading to IFN- production in pDCs. Then, the IFN- produced in pDCs activates the monocytes. Finally, the activated monocytes produce interleukin (IL)-18 and tumor necrosis factor (TNF)-, leading to activation of MAIT cells and induction of antigen-specific CTLs. On the other hand, the same study found that a conventional Ad5 vector failed to infect pDCs. TNF- production from peripheral blood mononuclear cells (PBMCs) following treatment with an Ad5 vector was lower than that with ChAdOx1.

High seroprevalence to Ad5 in adults due to the natural infection with an Ad5 has been reported in many studies [51] [52] [53] [54] [55] . Indeed, more than 80% of adults possess anti-Ad5 antibodies. Neutralizing anti-Ad5 In order to circumvent neutralizing anti-Ad5 antibody-mediated inhibition, other approaches have been

reported. An Ad vector coated with polyethylene glycol (PEG) can escape from neutralizing anti-Ad5 antibody-mediated inhibition by blocking the binding of anti-Ad5 antibodies to the virion [67] [68] [69] . Liposomeencapsulated Ad vectors have also been demonstrated to escape from neutralizing anti-Ad5 antibodies [70] [71] [72] .

Both cationic and anionic liposomes have been used for encapsulation of an Ad vector. In particular, because Ad vectors have a negative surface charge, they can be easily encapsulated by cationic liposomes simply by mixing the Ad vectors and cationic liposomes together [71] . Furthermore, recently, extracellular vesicles, such as exosomes, containing an Ad particle have recently been reported, although it remains unclear whether extracellular vesicles containing an Ad vector can circumvent the neutralizing anti-Ad5 antibodies [73] [74] [75] .

Both the PEG-modified and vesicle-encapsulated Ad vectors have the advantage that they can be repeatedly administered.

J o u r n a l P r e -p r o o f date, Ad vector vaccines for HIV have been eagerly pursued. Ad5 vector vaccines expressing HIV-1 gag/pol/nef (a 1:1:1 mixture of Ad vectors expressing each HIV-1 antigen) were tested in a double-blinded, randomized, placebo-controlled clinical trial [76, 77] . Although these Ad vector vaccines induced HIV-1specific CD8 + and CD4 + cell responses, the vaccine group tended to exhibit higher incidence of HIV-1 infection than the control group was found. In addition, risk factors for HIV acquisition would include pre-existing immunity to Ad5 in the vaccinated group [78] . In order to circumvent such pre-existing anti-Ad5 immunity, Ad vector vaccines based on Ad serotype 26 and chimpanzee Ad for HIV-1 have been tested in clinical trials [79, 80] . [81] , developmental research of Zika virus vaccines has been actively ongoing. For vaccination against Zika virus infection, genes encoding the membrane protein and envelop protein were incorporated into the Ad vector genome [82, 83] . An Ad serotype 26-based vector vaccine elicited high levels of neutralizing anti-Zika virus antibodies without severe side effects in a clinical trial [84] .

vaccines, inactivated virus vaccines, and split virus vaccines, are widely available, the immunization efficiencies of these vaccines are relatively low [85] . In addition, conventional influenza virus vaccines are strain-specific and have a very narrow range of coverage. Since there is a global concern that emerging influenza viruses, including avian influenza virus, have the potential to cause a pandemic, a novel platform of influenza virus vaccines is being developed. A replication-incompetent Ad5 vector expressing hemagglutinin (HA) and chimpanzee Ad-based vector (ChAdOx1) expressing nucleoprotein (NP) and matrix protein-1 (M1) have been tested in clinical trials [86, 87] . Furthermore, a replication-competent Ad serotype 4 (Ad4) containing the HA protein expression cassette in the E3 region showed prolonged systemic and mucosal immunity [88] . Replicating Ad4 has been orally administered to more than 10 million people as a vaccine against Ad4 respiratory disease and has shown no severe side effects [89] . Not only replication-incompetent but also replication-competent Ads would be a promising platform for Ad vector vaccines.

Ebola virus vaccine. Ebola virus disease causes severe symptoms, including high fever, headache, and diarrhea, with high mortality [90] . Ebola virus was first discovered in 1976 [91] , and since its discovery, there have been [96, 97] . Ad26.COV2-S also showed efficient vaccine efficacy (66.1%) [98] . More than 90% seroconversion rates were reported in the randomized controlled clinical trial of Ad5-nCOV [99] . However, ChAdOx1 nCoV-19 and Ad26.COV2-S have been reported to carry a potential risk of inducing thrombotic thrombocytopenia and disseminated intravascular coagulation, particularly in young adults, although the incident rates for these complications are very low [100] [101] [102] . Currently, further examination is actively underway to circumvent these side reactions. J o u r n a l P r e -p r o o f

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We would like to thank Aoi Shiota (Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan) for helpful discussion. This work was supported by JSPS/KAKENHI grant number 20H00664 and by a grant from the Japan Agency for Medical Research and Development (AMED) under Grant Number 20fk0108543h0001.

Authors declare no conflict of interest.