Recombinant Adenovirus Vectors as Mucosal Vaccines

C H A P T E R

24 Recombinant Adenovirus Vectors as Mucosal Vaccines Kristel L. Emmer1,2 and Hildegund C.J. Ertl2 1

Gene Therapy & Vaccines Program, University of Pennsylvania School of Medicine, Philadelphia, PA, United States 2Wistar Institute Vaccine Center, Philadelphia, PA, United States

I. INTRODUCTION Vaccines have saved millions of lives through the prevention of infectious diseases. Traditionally, most vaccines have been created by attenuation or inactivation of viral or bacterial pathogens. Advances in molecular virology have led to the development of new vaccine prototypes based on recombinant viral vectors that encode or express antigens from heterologous pathogens. Numerous viruses, including RNA and DNA viruses, have been modified to allow for their use as antigen-delivery vehicles [1
8]. Of those, vectors based on adenoviruses have consistently shown induction of potent and sustained B and T cell responses to inserted foreign antigens [9
12]. Replication-defective adenovirus vectors mostly based on human serotype 5 (HAdV5) were initially generated for the treatment of genetic diseases such as cystic fibrosis, hemophilia, and muscular dystrophy [13
15]. The recombinant adenoviruses had excellent transduction rates upon application through various

Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00024-9

routes. Nevertheless, expression of the therapeutic protein was transient, owing to robust immune responses directed toward both the adenoviral proteins and the transgene products causing rapid elimination of the infected cells [16,17]. Although the high immunogenicity of adenovirus vectors caused insurmountable problems for their use in permanent gene replacement protocols, it invited their use as vaccine carriers. Vaccine vectors have been generated from multiple adenovirus serotypes derived from different species. The vectors’ ability to replicate has been genetically altered. The foreign antigen has been expressed as a transgene, or epitopes thereof have been incorporated into the vectors’ capsid [18,19]. Methods for mass production, quality control, purification, and storage at ambient temperatures have been developed and are being refined [20
25]. Adenovirus vectors have been tested extensively in experimental animals as well as in human volunteers. In most human trials, vectors were given intramuscularly, but mucosal routes have been explored as well.

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© 2020 Elsevier Inc. All rights reserved.

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24. RECOMBINANT ADENOVIRUS VECTORS AS MUCOSAL VACCINES

Mucosal immunization has a number of advantages over systemic immunization. The relative ease of mucosal administration facilitates mass vaccination campaigns. It may reduce the cost of vaccination and decrease the risk of side effects due to inadvertent syringe or vial contamination. Since mucosal surfaces are the most common portal of entry for pathogens, vaccine-induced responses of the mucosal immune system, which is unique from the systemic immune system, may be better suited for prophylaxis. This review gives a brief overview about adenoviruses and adenovirus vectors and then discusses the use of adenovirus vectors for mucosal immunization, addressing available results, feasibility, and potential advantages as well as disadvantages.

II. ADENOVIRUSES Adenoviruses are nonenveloped viruses with an icosahedral capsid and a doublestranded DNA that ranges in size from 26 to 46 kb [26]. A number of early (E) and late (L) transcription units encode between 23 and 46 proteins. Human adenoviruses have the five early transcription units—E1A, E1B, E2, E3, and E4—which produce polypeptides that are required for viral transcription and replication of the viral DNA or serve to suppress the hosts’ immune responses. E1, E2, and E4 are essential, and their deletion cripples the ability of adenovirus to replicate. E3, which encodes polypeptides that block major histocompatibility class (MHC) I-restricted antigen presentation and inhibit various pathways of apoptosis, is nonessential for viral replication. The five late transcription units L1
L5 encode the three major capsid proteins, that is, hexon, fiber, and penton base, as well as a number of minor capsid components that stabilize the capsid or participate in its assembly.

Adenoviruses are species-specific and infect a wide range of hosts, including mammals, birds, and amphibians. Adenoviruses are divided into five genera. The genus Mastadenovirus includes all of the human adenoviruses, which are subdivided into seven species, A
G. Each species is further separated into serotypes, which are distinguished by their reactivity with virus-neutralizing antibodies directed mainly to the hypervariable loops of the major coat protein hexon. Neutralizing antibodies can also be directed against fiber, although this response is less pronounced. To date, 57 distinct serotypes of human adenoviruses have been identified. Adenoviruses cause upper respiratory infections, conjunctivitis, tonsillitis, or otitis. Human serotypes 40 and 41 cause gastroenteritis. Infections are usually self-limiting, but can be fatal in immunocompromised individuals or occasionally in otherwise healthy humans. Adenovirus vaccines are not available to the general public; however, vaccines to serotypes 4 and 7 are given to U.S. military recruits. Adenoviruses persist even though their genome does not integrate into the host cell genome. The level of persistence seems to depend on the serotype and the host species. While most humans stop shedding adenovirus within a few weeks after infection, adenoviruses can readily be isolated from the feces of most nonhuman primates [27]. Even after cessation of shedding, the virus continues to persist episomally at low levels in T cells, presumably for the lifetime of the individual [28]. The continued presence of adenovirus in turn maintains an effector-like T cell response to its antigens [29,30].

III. IMMUNE RESPONSES TO ADENOVIRUSES Adenoviruses induce vigorous innate and adaptive immune responses. Innate responses

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

III. IMMUNE RESPONSES TO ADENOVIRUSES

are triggered by a number of pathogenassociated molecular patterns (PAMPs) on adenoviruses interacting with host cell pathogenrecognition receptors (PRRs), which are located on the cell surface, in endosomes, and in the cytosol. Adenoviruses bind to cellular receptors such as the coxsackievirus and adenovirus receptor (CAR) or integrins, used by most human serotypes, and are then are taken up by endocytosis [31]. Cytokines such as interleukin 8 (IL-8) or tumor necrosis factor alpha (TNF-α) facilitate entry by increasing the density of CAR and integrins on the cell surface [32]. Species B adenoviruses bind to CD46 or desmoglein-2 and are taken up by pinocytosis [33]. On the cell surface, adenoviruses interact with toll-like receptor 2 (TLR-2), triggering a proinflammatory response [34]. Adenoviruses that form a complex with coagulation factor X can also interact with TLR-4, another cell surface PRR [35]. CD46-binding adenoviruses have been reported to interact with TLR-9 [36], a sensor for unmethylated CpG motifs in double-stranded DNA [36]. Within the cytosol, the adenoviral genome binds to additional sets of DNA sensors such as NLRs, which are the core proteins of inflammasomes, gammainterferon-inducible protein 16 (IFI16), DEADbox helicase 41 (DDX41), and protein cyclic GMP-AMP synthase (cGAS); the latter initiates the stimulator of interferon genes (STING) pathway [37]. The adenovirus genome encodes two short viral RNAs that are produced with the help of host cell polymerases and interact with RIG-I, a cellular double-stranded RNA sensor. These different PAMP
PRR interactions trigger a robust proinflammatory response to adenoviruses, which is also observed with adenoviral vectors [38]. The strong innate responses facilitate induction of adaptive responses by providing a proinflammatory milieu rich in cytokines and activated professional antigen-presenting cells. B cells are induced to different late gene products and produce both nonneutralizing and

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neutralizing antibodies. Nonneutralizing antibodies directed to different adenovirus proteins cross-react between serotypes. Neutralizing antibodies, directed against hexon or fiber, are serotype-specific. The prevalence of neutralizing antibodies to adenoviruses differs depending on serotypes and geographic location [39
41]. In the United States, there is a clear distinction between common serotypes to which a large proportion of the adult population carries neutralizing antibodies, best exemplified by adenovirus of human serotype 5 (HAdV5), and so-called rate serotypes to which fewer than 10% of human adults show serological evidence of preexisting immunity. This distinction is blurred in less developed countries, where neutralizing antibodies to serotypes that are rare in the United States tend to be far more common, as best exemplified by HAdV26 (Table 24.1). Preexisting neutralizing antibodies induced by natural infection lessen the uptake of adenoviral vectors. This in turn diminishes the amount of transgene that is synthesized, and thereby reduces immune responses to the vaccine antigen [42
44]. Neutralizing antibodies to simian origin adenoviruses, which are phylogenetically clustered with human adenoviruses in the genus Mastadenovirus, are rare in humans [39,45]. Vectors based on nonhuman primate serotypes have been developed as vaccine carriers to circumvent preexisting immunity to human serotypes and therefore avoid dampening of transgene product-specific immune responses. Adenoviruses induce both CD41 and CD81 T cell responses to E and L gene products. These T cell responses cross-react between multiple serotypes [46]. Owing to the low-level persistence of adenoviruses, ratios of T cells to adenoviral antigens are fairly high in humans. On average, 1%
2% of CD41 and 3%
5% of CD81 T cells in human blood respond to adenovirus antigens. T cells produce different effector functions, including interferon gamma (IFNγ), TNF-α, and lytic enzymes [46].

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24. RECOMBINANT ADENOVIRUS VECTORS AS MUCOSAL VACCINES

TABLE 24.1

Prevalence of Adenovirus Neutralizing Antibodies in Humans (Percentage of Population)

Serotypes

United States (%)

Europe (%)

South America (%)

Africa (%)

Asia (%)

HAdV5

40
70

60

80

80
90

70
90

HAdV6

50

50

50

70
80

70
80

HAdV26

, 10

90

50

90

20
50

HAdV35

, 10

HAdV36

, 10

HAdV41

, 10

SAdV23

, 10

SAdV24

, 10

IV. TYPES OF ADENOVIRUS VECTORS Replication-competent adenovirus (RCA) vectors that are deleted only in E3 have been constructed. Such vectors may not meet the safety requirements for human preventive vaccines; however, they have been used for immunization of wildlife against rabies [47,48]. Most adenovirus vectors are deleted in E1, which is essential for viral replication, as it encodes polypeptides that initiate transcription of viral genes. E1 deletions thus render adenoviruses replication-defective. E1-deleted adenovirus vectors can be grown in cell lines such as HEK 293 cells or PerC6 cells that transcomplement E1. For many serotypes, including simian adenoviruses (SAdV), the deleted E1 domain can be transcomplemented by E1 of HAdV5; for others, packaging cell lines carrying the serotype-specific E1 have to be developed. Many adenovirus vectors are deleted in both E1 and E3 to increase the permitted size of the transgene’s expression cassette that is generally inserted into E1. For expression of toxic glycoproteins, partial E3 deletions that maintain sequences encoding antiapoptotic peptides have been explored [49]. Additional deletions, such as deletion of E4, have been used to

17
20 50

50

50 50

20

5
30

5
12

5
10

13

dampen immune responses against antigens of adenovirus, and again these genes have to be provided in trans during virus propagation [50]. Some vectors based on rare or nonhuman primate serotypes are further modified to replace part of the E4 domain with that of HAdV5 to increase vector yields during production [51]. Vectors that express two expression cassettes in forward or reverse orientation within E1 and E3 have been developed [52]. Expression of the transgene product is in general driven by a potent ubiquitously active promoter such as the early promoter of cytomegalovirus combined with an enhancer and introns to optimize protein production. Instead of using adenovirus vectors as genedelivery vehicles, other researchers have modified the viral hexon by incorporating short, linear B cell epitopes from a different virus [18,19,53]. Such vectors, which display the epitopes in a repetitive fashion on the virus surface, promote the induction of B cell responses and could be combined with second, longer sequences incorporated within an expression cassette into E1. Adenovirus vectors have been generated from multiple different human and nonhuman serotypes. Although many of the characteristics of such vectors are similar, there are also

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

VI. QUALITY CONTROL OF ADENOVIRUS VECTORS

notable differences in their performance as vaccines, as discussed below.

V. CONSTRUCTION, PURIFICATION, AND TITRATION OF ADENOVIRUS VECTORS Initially, adenovirus vectors were constructed by homologous recombination. This rather cumbersome procedure has been replaced by using viral molecular clones in which the adenovirus sequences with appropriate deletions and suitable rare restriction enzymes sites flanking the deletions are cloned into a plasmid vector or a bacterial artificial chromosome for propagation in bacteria [54,55]. By using standard cloning procedures, the transgene expression cassette is first cloned into a pShuttle vector and, from there, is inserted into the deleted E1 domain of the viral molecular clone. The recombinant viral molecular clones are linearized by a restriction enzyme targeting a site just upstream of the 50 inverted terminal repeat of the adenovirus genome and transfected into packaging cells. The recombinant virus, which in general forms plaques on the packaging cell monolayers within 7
14 days, is then serially expanded. Once a sufficiently large batch has been generated, the adenovirus vector is released by freeze-thawing or by treatment with detergents. The vector is then cleared by low-speed centrifugation followed by cesium chloride gradient purification. Alternative purification methods that avoid centrifugations are available; these include clearance by filtration and purification by chromatography [23
25,56]. The viral particle content of a vector preparation is determined by its absorbency at 260 nm. In clinical trials, adenovirus vectors are dosed according to their viral particle content, as this parameter directly determines the vector’s toxicity. Immunogenicity of the vectors, on the other hand, depends on the

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number of virus particles that are able to infect cells and to transcribe the transgene product. In general, the numbers of infectious virus particles are measured by plaque assays or by an endpoint dilution assay for cytopathic effects. Plaque formation is dependent on several viral factors and is not reliable for all adenovirus serotypes. Alternative assays based on amplification of the adenovirus vectors transcripts or on staining for hexon with an antibody to a conserved domain of adenovirus hexon have been developed and validated.

VI. QUALITY CONTROL OF ADENOVIRUS VECTORS The numbers of release assays that are required for adenovirus vectors used in clinical trials is extensive and beyond the scope of this article. For preclinical experiments, vector batches are checked for RCA, which can emerge during the creation and propagation of E1-deleted replication-defective adenoviruses as a result of recombination between overlapping viral sequences in the packaging cells and the vectors [57]. Depending on the level of RCA in vector preparations, it can have a significant impact on vector performance, host immune responses, and toxicological profiles for in vivo experiments. Vectors should be tested for sterility and endotoxin content. The genetic integrity and stability of vectors should be assessed by restriction enzyme digest of purified viral DNA from early passaged virus and virus that has been propagated sequentially for 12
15 passages in comparison to the viral molecular clone. Although most adenovirus vectors are genetically stable, deletion of the transgene or part of the backbone can happen and may necessitate rerescue of the virus or even reconstruction of the viral molecular clone.

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

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24. RECOMBINANT ADENOVIRUS VECTORS AS MUCOSAL VACCINES

VII. THERMOSTABILITY OF ADENOVIRUS VECTORS Adenovirus vectors kept in suitable buffers are stable if stored at temperatures below 65 C. Upon storage at room temperature, they rapidly lose infectivity. Cold chains are difficult to maintain in less developed countries, and methods have been developed to increase the thermostability of adenoviral vectors [58,59]. These methods rely on resuspension of vectors in solutions with a high sugar content, such as mannitol and sucrose or trehalose and sucrose, followed by spray-drying or dry-coating onto membranes. Dried vectors in sugar formulations are stable for weeks at room temperature.

VIII. IMMUNOGENICITY OF ADENOVIRUS VECTORS E1-deleted adenovirus vectors induce potent transgene product-specific T and B cell responses after a single dose. Upon systemic immunization, CD41 T cell responses are biased toward T helper 1 (Th1) cell responses that support induction of cellular immunity [58,59]. Immune responses are sustained, presumably owing to low-level vector persistence in a transcriptionally active form within lymphatic tissues [29,30]. Despite continuing T cell stimulation, adenovirus vector-induced T cells do not differentiate toward exhaustion, but rather maintain populations of effector and effector memory cells. Immune responses can be further increased by booster immunizations using an adenovirus vector from a different serotype or an unrelated vaccine prototype [60
62]. Although all adenovirus vectors tested to date have shown excellent immunogenicity, there are some clear differences. Innate responses to some of the chimpanzee adenovirus vectors derived from species E were shown to be more pronounced than those to HAdV5 [63]. Another study described that vectors

based on CD46-binding adenoviruses of species B elicit higher levels of proinflammatory cytokines compared to HAdV5 vectors, a species C member [64]. Vectors based on species C viruses tend to induce more potent adaptive immune responses than those from species E viruses, which in turn, are more immunogenic compared to B virus vectors [65]. This may, in part, relate to the hierarchy of proinflammatory responses which, when overly induced, may dampen adaptive responses [66].

IX. THE MUCOSAL IMMUNE SYSTEM Mucosal surfaces that cover the gastrointestinal, urogenital, and respiratory tracts as well as the eyes, middle ears, and exocrine gland ducts are the most common portals of entry for pathogens. In addition, foreign innocuous antigens from food or commensal bacteria constantly bombard mucosal surfaces. The mucosal immune system must therefore distinguish between harmless antigens and those derived from pathogens, a challenge that is not faced by the systemic immune system. The mucosal immune system is thus distinct yet interconnected with the systemic immune system [67]. The mucosal immune system is divided into inductive sites, which are the organized mucosaassociated lymphoid tissues, and effector sites, which are lymphocytes dispersed within mucosal membranes. Inductive sites such as Peyer’s patches in the small intestine also include lymph nodes draining the mucosal surfaces, such as mesenteric lymph nodes within the peritoneal cavity or the Waldeyer’s tonsillar ring of the nasopharynx and oropharynx. Inductive sites contain B cell follicles with follicular dendritic cells, which are surrounded by T cells. A single layer of epithelial cells separates the lymphoid tissue from the mucosal surface. Within the gut, this area contains microfold (M) cells, which take up antigen from the lumen and then transfer it to antigen-presenting cells. Other

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X. MUCOSAL VACCINES

mechanisms allow mucosal dendritic cells to take up antigen. Soluble antigen crosses epithelial barriers. Larger particulate antigen is transferred across epithelial cells through transcellular routes by enterocytes and potentially by macrophages and CX3CR11 myeloid cells. MHC class II positive enterocytes release partially degraded antigen in the form of exosomes. The antigen is then taken up by dendritic cells within the lamina propria and transported to inductive sites. Mucosal dendritic cells are distinct from those present in the central immune system, presumably owing to differences in their microenvironment, which, for example, within the gut is rich in retinoid acids (RAs). RAs prime dendritic cells to induce IL-10 and transforming growth factor beta (TGF-β), resulting in the generation of regulatory T cells (Tregs) [68], which play a crucial role in maintaining tolerance to harmless antigens (Chapter 3: Mucosal Antigen Sampling Across the Villus Epithelium by Epithelial and Myeloid Cells. Chapter 28: M Cell-Targeted Vaccines). Effector sites such as the lamina propria and the interstitial epithelium of the gut mucosa contain intraepithelial lymphocytes (IELs) that originate either directly from the thymus (tIELs) or from the periphery (pIELs). tIELs are largely T cell receptor (TcR)γδ1CD8αα1 or TcRα/β1CD8αα1). They migrate from the thymus to the gut shortly after birth, while pIELs, which are TcRαβ1CD81 or TcRαβ1CD41, are induced in peripheral lymphatic tissues and then migrate to the mucosa. Within the mucosa, pIELs adapt and progressively increase expression of CD8αα homodimers [69]. Mucosal B cells belong both to the conventional B2 subset and the B1 subsets; the latter may arise from peritoneal B1 cells. Mucosal B cells mainly produce secretory immunoglobulin A (SIgA), which is resistant to degradation, and thus is able to survive the harsh environment of the gastrointestinal tract. Class-switch recombination of mucosal B cells is facilitated by local follicular Th cells, which originate from Th17 cells and, through

the secretion of IL-21 and TGF-β, promote class switching to IgA [70]. Class switching can also be achieved without T cell help through direct interaction of the B cells’ TLRs with PAMPs on gut bacteria [71] or by microbiota metabolites [72]. The common mucosal immune system is partially interconnected in that induction of a response at one site leads to effector responses at distant sites. Nevertheless, responses are compartmentalized in that oral immunization through the gastrointestinal tract favors local responses; intranasal immunization favors responses within the oral cavity, the airways, and the female reproductive tract; and intrarectal immunization induces the strongest responses within the rectum and the genital tract but not within the oral cavity. Vaginal immunization induces a weak and mainly local response, reflecting the lack of local inductive sites in the vaginal mucosa. The mucosal immune system is shaped and regulated by the microbiome and vice versa: The composition of the microbiome is molded by the mucosal immune system [73] (Chapter 9: Influence of Commensal Microbiota and Metabolite for Mucosal Immunity). The microbiome varies between individuals, in part owing to differences in diets, and may thus influence the outcome of mucosal immune responses to pathogens or vaccines. This clearly has to be investigated in more depth, as geographic differences in the microbiome may make it difficult to predict global mucosal vaccine efficacy based on clinical trials, which are typically conducted in a few selected areas.

X. MUCOSAL VACCINES Most commercially available vaccines are administered by a systemic route such as intramuscular, subcutaneous, or intradermal injections. Only six vaccines used in humans are given to mucosal surfaces. Most are live attenuated vaccines such as those for poliovirus,

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

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24. RECOMBINANT ADENOVIRUS VECTORS AS MUCOSAL VACCINES

rotavirus (see Chapter 40: The Role of Innate Immunity in Regulating Rotavirus Replication, Pathogenesis, and Host Range Restriction and the Implications for Live Rotaviral Vaccine Development and Chapter 41: Development of Oral Rotavirus and Norovirus Vaccines), Salmonella enterica serovar Typhi (see Chapter 29: Induction of Local and Systemic Immunity by Salmonella Typhi in Humans), Vibrio cholerae (see Chapter 31: Cholera Immunity and Development and Use of Oral Cholera Vaccines for Disease Control), adenovirus serotypes 4 and 7 given orally, and a coldadapted influenza vaccine given intranasally (see Chapter 39: Nasal Influenza Vaccines). Several rabies vaccines based on a recombinant vaccinia virus vector, a recombinant replicationcompetent HAdV5 vector expressing both rabies virus glycoproteins as their transgene product, or attenuated rabies viruses have been used for oral immunization of wildlife. With the exception of the rabies and adenovirus vaccines, mucosal vaccines are given through their natural routes of infection, which avoids the need to protect the vaccine from digestion during passage through the gastrointestinal tract or to facilitate uptake through the mucosa. Oral rabies vaccines virus do not pass into the intestinal tract, but rather induce a response locally within the oral cavity. The vaccines for adenovirus serotypes 4 and 7 induce immune responses within the intestine. They are given in tablets with an inner capsule containing dried virus and an outer layer containing microcrystalline cellulose, magnesium stearate, and anhydrous lactose and an enteric coating consisting of cellulose acetate phthalate, alcohol, acetone, and castor oil, which protects the virus until it has passed through the upper digestive tract [74]. Immunity induced by live attenuated vaccines given systemically, such as the yellow fever vaccine, is very long lasting [75]. Less is known about the longevity of local immune responses induced by mucosal vaccinations. Available evidence obtained upon oral application of poliovirus vaccines suggests that responses may

wane after a fairly short period (approximately 1 year) [76]. Age may also affect the effectiveness of mucosal vaccines, presumably in part owing to the delayed development of the mucosal immune system. The killed oral cholera vaccine, for example, induces a CD41 T cell response in older children, but is relatively ineffective in children under 6 years of age [77]. A study conducted with the pentavalent rotavirus vaccine in Nicaragua reported good protection during the first year after vaccination but a decline in prevention after that [78]. In contrast, an inactivated whole cell oral cholera vaccine or an attenuated S. Typhi live oral vaccine resulted in sustained protective immunity for a 3-year or a 7-year period, respectively [79].

XI. ADENOVIRUS VECTORS AS ORAL VACCINES Oral immunization is the method of choice for vaccine delivery. It is less expansive, it does not require the extensive purification needed for systemic vaccines, mass vaccination programs can be conducted rapidly and with ease, and it would be safe for replication-defective adenovirus vectors. Replicating adenoviruses would be less suited for use in humans, as the viruses would be shed and then pose risks of fecal transmission to others. As an additional benefit, preexisting neutralizing antibodies to adenovirus, which dampen transgene product-specific immune responses upon systemic immunization, fail to affect those induced by oral immunization. Most adenovirus serotypes infect through the mucosal membranes of the airways, the naso-oropharynx, or the conjunctiva. Only two serotypes, HAdV40 and HAdV41, belonging to species F, are enteric viruses. Nevertheless, some humans and nearly all great apes shed multiple serotypes of infectious adenoviruses, indicating that these viruses survive within the intestinal tract [28,80]. By the same token, DNA from most adenovirus species has been isolated from intestinal tissues of primates [81].

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

TABLE 24.2 Oral Adenovirus Vector Vaccines

Vector (s)

Regimen

Species

Transgene product

Vaccine target

Immune responses/protection/side effects

Interference by vectorspecific NAs

References

REPLICATION-COMPETENT HAdV7, Prime-boost HAdV4

Chimpanzee, Hepatitis B virus dogs (HBV)

Surface antigen Antibody responses, partial protection (sAg)

n.t.

[82,83]

HAdV7, Prime-boost HAdV4

Humans

HBV

sAg

No response

n.t.

[84]

CAdV2

1 dose

Raccoons

Rabies virus

Glycoprotein (G)

Multifocal necrotizng bronchiolitis

n.t.

[85]

HAdV5

1 dose

Mice

Human Gag immunodeficiency virus (HIV)-1

B and T cells

n.t.

[86]

CAdV2

1 dose

Cats

Rabies virus

G

Poorly immunogenic per os, protective after systemic immunization

n.t.

[87]

HAdV5

1 dose

Raccoons, skunks

Rabies virus

G

Protective levels of serum antibdy titers

n.t.

[88]

CAdV2

1 dose

Raccoons, skunks

Rabies virus

G

Neutralizing antibodies, protection against challenge

n.t.

[89]

HAdV4

Enteric-coated capsules, 3 doses 6 parental H5N1 vaccine boost

Humans

Influenza virus

Hemagglutinin (HA)

Cellular responses, low seroconversion rates after oral immunization, increase after parental boost, oral priming increases antibody avidity

Yes at lower vaccine doses

[90,91]

REPLICATION-DEFECTIVE HAdV5

1 dose

Mice, cotton rats

Measles virus

HA, fusion (F) protein

Antibodies, protection

n.t.

[92]

HAdV5

1 dose

Mice

Measles virus

Nucleocapsid protein (NC)

Antibodies, splenic CD81 T cells in spleens

n.t.

[93]

HAdV5, 1 dose SAdV25

Mice

Rabies virus

G

Antibodies in serum, vaginal lavage, feces, protection, biodistribution mainly to oral cavity and airways

no

[94,95]

HAdV5, 1 dose SAdV25

Neonatal mice

Rabies virus

G

Antibodies in serum, vaginal lavage, feces, protection

n.t.

[96] (Continued)

TABLE 24.2 (Continued)

Vector (s)

Regimen

Species

Vaccine target

Transgene product

Immune responses/protection/side effects 1

Interference by vectorspecific NAs

References

SAdV23

1 dose, prime-boost

Mice

HIV-1

Gag

CD8 T cells, protection against surrogate challenge

n.t.

[97]

SADv?

1 dose

Mice

HIV-1

Gag

Intestinal CD81 T cells induced by IM but not oral immunization

n.t.

[98]

HAdV5

1 dose

Mice

Ebola virus

G

Systemic and mucosal T and B cell responses, protection

n.t.

[99]

HAdV5

1 enteric-coated dose Macaques

HIV-1

Gag, Env epitopes

T cell responses

n.t.

[100]

HAdV5

1 dose

Mice

respiratory syncytial virus (RSV)

G

No response after oral immunization

n.t.

[101]

HAdV41 HAdV5 boost

Mice

HIV-1

Env

Intestinal CD81 T cell response after oral n.t. immunization and HAdV5 boost, more potent after ileal priming

[102]

HAdV5

1 dose, iliac immunization, HAdV5 i.m. boost

Mice

HIV-1

Env

Intestinal CD81 T cell responses after iliac immunization, increase after boost

n.t.

[103]

HAdV5

1 dose, sublingual

Mice

HIV-1

Gag

Systemic and mucosal CD81 T cells

no

[104]

HAdV5

1 dose, adjuvant

Mice, ferrets

Influenza virus

HA

Antibodies, protection

no

[105]

HAdV5

1 dose

Mice

C. botulinum

Heavy-chain C- Antibodies, protection fragment

n.t.

[106]

HAdV5

1 or 2 doses, encapsidated, adjuvant

Humans

Avian influenza virus

HA

CD81 T cell responses

n.t.

[107]

HAdV5

1 or 2 doses, encapsidated, adjuvant

Humans

Influenza virus

HA

Increases in neutralizing antibody titers

No

[108]

HAdV5

1 or 2 doses, radiocontrolled capsules, adjuvant

Humans

Influenza virus

HA

Mucosal and serum B cell responses, higher upon vaccine release in the ilium than the jejunum

n.t.

[109]

n.t., not tested.

XI. ADENOVIRUS VECTORS AS ORAL VACCINES

Oral vaccine studies have been conducted with both replication-competent and replicationdefective adenovirus vectors derived from different human and nonhuman serotypes [82
101] (Table 24.2). Although poorly immunogenic in cats [86], RCA vectors, based on a canine serotype expressing the rabies virus glycoproteins, were shown to induce neutralizing antibodies and protect against challenge in raccoons and skunks [87,88]. Vectors based on human serotype 4 or 7 induced immune responses, albeit low ones, to hepatitis B surface antigen (HBsAg) in chimpanzees [82]. A clinical trial was conducted with the replication-competent HAdV7-HBsAg vector [84]. The vaccine was given to three human volunteers in the form of enteric-coated tablets. The vaccine was well tolerated and was shed for up to 2 weeks. Two out of three vaccinees showed increases in antibody titers to HAdV7, and none of them developed antibodies to hepatitis B virus. Another clinical trial was conducted using the live HAdV4 vector expressing the hemagglutinin of the poorly immunogenic H5N1 virus [90,91]. Each volunteer received three doses of vector in enteric-coated capsules. Some were then boosted parentally with an inactivated H5N1 vaccine. The oral HAdV4 vaccine-induced cellular immune responses, but only marginal neutralized antibody titers. Nevertheless, upon a systemic boost with a traditional vaccine, titers and avidity of influenza virus-specific antibodies were markedly higher in HAdV4-immunized individuals than in volunteers who had received only the systemic vaccine, indicating that the oral vaccine had primed the B cell compartment. Several preclinical studies report on oral immunizations with replication-defective adenovirus vectors. Oral immunization with HAdV5 or a SAdV of species E expressing the highly immunogenic rabies virus glycoprotein was shown to induce antibody responses to the transgene product in mice [94,95]. Using a different transgene in another SAdV serotype for oral immunization of mice resulted in genital CD81 T cell responses [97]. Additional studies

429

showed that T cell responses within the gut or the female genital tract were more robust upon systemic delivery than mucosal vaccine delivery [98]. Biodistribution studies in mice showed that, upon oral immunization, the adenovirus vectors were mainly recovered from cells of the oral cavity and the airways but not from the intestine [94]. Another group used a HAdV5 vector backbone for gastric immunization of mice to a measles virus antigen and reported induction of sustained humoral and cellular responses, in most, but not all mice [93]. Direct surgical inoculation of HAdV5 vectors into the intestine resulted in potent immune responses in experimental animals that were not achieved upon oral immunization [103]. Our attempt to induce antibody responses failed in nonhuman primates following oral immunization with high doses of SAdV vectors that were found to be efficacious in orally immunized mice (unpublished data). Oral immunization with nonenteric adenovirus vectors, when applied in a liquid form rather than within enteric-coated capsules, induced immune responses locally within the oral cavity and the airways rather than within the intestinal tract as a consequence of the vectors being labile to the stomach’s low pH and to gastric and pancreatic enzymes [103]. This is further supported by a study that reported that in mice, an E1-deleted HAdV5 vector induces higher immune responses upon sublingual than oral application [104]. While oral adenovirus vector immunization of mice has repeatedly been reported to induce transgene productspecific immune responses, oral immunization of nonhuman primates using the same vectors failed. This could reflect anatomic differences within the oral cavity. Primates have tonsils and adenoids guarding the oral cavity and the pharynx, while most rodents lack these organized structures, but instead have more diffuse nasal-associated lymphoid tissues that may more readily be infected with adenovirus vectors. Chewing habits differ between species. Raccoons and skunks chew their food carefully,

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

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24. RECOMBINANT ADENOVIRUS VECTORS AS MUCOSAL VACCINES

allowing for their oral vaccination with recombinant vaccinia or adenovirus vectors to rabies, while dogs swallow big chunks of food and do not respond as well to oral immunization with the same vaccine virus vectors. If the duration of contact between the recombinant vaccine vector and the mucosa of the oral cavity indeed affects effectiveness of oral immunization, it would be unsuited for small children, as they may or may not follow instructions. Vectors based on nonenteric adenoviruses could be encapsulated similarly to the live HAdV4 and HAdV7 vaccines used by the U.S. military. To this end, an enteric polymethacrylate formulation for coating hydroxy-propylmethyl-cellulose capsules containing lyophilized HAdV5 expressing HIV-1 gag and a string of Env epitopes was used [100]. Vectors were thermolabile, and infectivity dropped within a week when tablets were stored at 4 C. Animals fed with these capsules developed transient SIgA responses, and responses in saliva were detected in only two thirds of animals. Cellular responses were weak but could be boosted by an adjuvanted Env peptide cocktail. Additional clinical trials tested the immunogenicity of an HAdV5 influenza vaccine given within enteric-coated tablets. The HAdV5 vectors expressed the virus hemagglutinin together with a molecular double-stranded RNA hairpin as a TLR adjuvant. The vaccine was well tolerated, and volunteers were shown to develop transgene product-specific CD81 T cells and antibodies. Immune responses were not affected by preexisting antibodies to the vaccine carrier [107,108]. In a follow-up trial, humans were given the HAdV5 vector within radio-controlled capsules that allowed for vaccine release within the ilium or the jejunum [109]. Systemic and mucosal immune responses were induced at either site, but rates of vaccine responders were higher when the vaccine was released within the ilium. These results are very promising and invite further trials. One of the attractive features of adenovirus vectors is that once their production has been

optimized, they are expected to be highly cost effective, with a single dose within the $1 range [110]. Encapsulation would increase the cost, which could be offset in part by reduced production cost due to less extensive purification and savings during vaccine delivery. Methods to ensure thermostability would have to be modified to allow for their use in encapsidated adenovirus vectors. Alternatively, one could develop vectors based on enteric adenovirus serotypes of species F. E1-deleted vectors based on HAdV41 have been constructed. The vector, which was genetically unstable [111], was shown upon oral immunization to induce a low immune response in mice that could be boosted with a HAdV5 vector expressing the same transgene and given systemically [102]. This line of research should be pursued but would first necessitate the development of genetically stable vectors.

XII. ADENOVIRUS VECTORS AS INTRANASAL VACCINES Adenovirus vectors transduce the endothelial cells covering the airways, although transduction is inefficient, as the major adenovirus receptor CAR is expressed on the basolateral rather than the apical surface of airway epithelial cells [112]. Nevertheless, a number of preclinical studies explored intranasal immunizations with adenovirus vectors derived from different serotypes [113
152] (Table 24.3). Initial studies used RCA vectors based on human, canine, or bovine serotypes expressing a variety of foreign viral antigens and reported induction of mucosal and systemic antibody responses, including IgA responses in mice, rats, dogs, cats, and nonhuman primates [86,113
122]. In mice, replication-competent HAdV5 vectors were shown to induce mucosal memory CD81 T cells [122]. Responses were blunted in the presence of preexisting immunity to the vaccine carrier [120].

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

TABLE 24.3 Vector(s)

Intranasal Adenovirus Vector Vaccines

Regimen

Species

Vaccine target

Transgene product

Immune responses/ protection

Interference by preexisting NAs

References

n.t.

[113]

REPLICATION-COMPETENT VECTORS HAdV4, 7, 5

Vectors given sequentially

Chimpanzee

HIV-1

Gag, Env

Serum, salivary, nasal, rectal, vaginal secretions, proliferative T cell responses

HAdV5

1 dose

Mice

Herpes simplex virus (HSV)-2

gB

IgA and IgG responses n.t. serum, vaginal secretions, transient systemic and sustained mucosal CD81 T cells, protection

[114,115]

HAdV5

1 dose

Mice

Simian Gag immunodeficiency virus (SIV)

n.t. Sustained systemic antibody and CD81 T cells

[86]

HAdV5

1 dose

Cotton rats, mice

Bovine herpes gD virus type (BHV) 1

IgA in lungs and nasal wash, systemic T cells, protection

n.t.

[116,117]

HAdV5

2 doses

Cattle

Foot and mouth disease virus

Precursor polypeptide 1

No protection

n.t.

[118]

BAdV3

1 dose

Cotton rats

Viral diarrhea virus

gE2

Systemic and mucosal IgA n.t. and IgG

[119]

CAdV2

1 dose

Canine puppies

Canine distemper virus

HA, F

Protection in CAdV2 seronegative but not seropositive puppies

Yes

[120]

CAdV2

1 dose

Mice

Rabies virus

Glycoprotein (G)

Serum neutralizing antibody titers, protection

n.t.

[121]

HAdV5

Listeria vector prime and protein boost

Macaques

SIV

Gag

T cells to Gag in blood and n.t. rectal mucosa, partial protection

[122]

REPLICATION-DEFECTIVE HAdV5

1 dose

Mice

Clostridium tetani

Tetanus toxin C fragment

Protection

n.t.

[123]

HAdV5

1 dose

Calves

BHV 1

gD or gC

Protection after second vaccine dose

n.t.

[124] (Continued)

TABLE 24.3

(Continued) Interference by preexisting NAs

References

n.t.

[125]

Gag, Pol, Env

Mucosal IgAs, Yes transduction of the central nervous system (CNS)

[126]

EGFP

Biodistribution study: transduction of the olfactory bulb but not other areas of the CNS

n.t.

[127]

Rabies virus

G

IgA in serum, genital secretions, and feces

n.t.

[128]

Mice

Rabies virus

G

Systemic and genital antibody responses, impaired B cell response in HAdV5-per-immune animals without DNA prime

Yes

[129]

1 dose

Neonatal mice

Rabies virus

G

Protection

n.t.

[96]

SAdV23

1 dose

Mice

HIV-1

Gag

Genital CD81 T cells

n.t.

[130]

HAdV5

1 dose

Neonatal mice

Rotavirus

VP4

intestinal IgG and IgA, immunization of dams protects offspring

n.t.

[131]

HAdV5

1 dose

Mice

MCMV

gH

Antibody responses in sera, bronchoalveolar lavage, fecal suspensions and vaginal washings; protection

n.t.

[132]

HAdV5

After BCG prime

Mice, pigs

Mycobacterium tuberculosis

Ag85a

Increased protection

n.t.

[133,134]

Vaccine target

Transgene product

Immune responses/ protection

Vector(s)

Regimen

Species

HAdV5

1 dose

Mice

Murine cytomegalovirus (MCMV)

gB

Antibodies in serum, bronchoalveolar lavage, fecal suspensions and vaginal washings, protection

HAdV5

1 dose

Mice

HIV-1

HAdV5

1 dose

Mice

HAdV5

1 dose

Mice

HAdV5

DNA vaccine prime

HAdV5,

HAdV5

1 dose

Mice

Norovirus

Capsid protein

IgG, IgA, IGM in sera, n.t. feces, intestines, lungs and lung lavage, systemic T cells

[135]

HAdV5

1 dose

Mice

RSV

G

Mucosal IgA and serum IgG responses, systemic CD81 T cell responses, protection

n.t.

[101,136]

HAdV5

1 dose

Mice

Clostridium botulinum

Heavy-chain C-fragment

Mucosal IgA and IgG, protection

No

[137]

HAdV5

1 dose

Mice

Streptococcus pneumoniae

sAg A, detoxified pneumolysin

Serum IgG, protection

n.t.

[138]

HAdV5

1 dose

Mice

Mycobacterium tuberculosis

Ag85a TB0:4

T cells in spleen and lung lumen, protection

n.t.

[139]

HAdV5, HAdV35

1 dose, aerosols

Mice, ferrets

Various

Various, including influenza virus HA

T cells in blood, BAL, protection against influenza virus challenge

n.t.

[140]

HAdV5

1 dose

Guinea pigs

Ebola virus

G

Protective immunity

no

[141]

HAdV5

1 dose

Mice

Hepatitis C virus (HCV)

CE1
E2

High antibody and low T cell responses, protection against surrogate challenge

n.t.

[142]

HAdV5

Protein boost

Mice

Chlamydia

CPAF

Strong antibody and weak n.t. T cell responses after the prime, protection after the boost

[143]

HAdV5

1 dose

Pigs

Influenza virus

HA

n.t. Full protection against homologous challenge, partial protection against heterologous challenge, vaccine-induced enhanced respiratory disease following heterologous challenge

[144]

(Continued)

TABLE 24.3

(Continued) Interference by preexisting NAs

References

Strong T and B cell responses systemically and within the airways, protection

n.t.

[145]

HA

Stronger neutralizing antibody responses than after IM immunization, protection

No

[146]

RSV

G, F

Weak serum antibody responses, IgG and IgA in BAL, protection

n.t.

[147]

Mice

Influenza virus

HA and ectodomain of M2

Serum antibody responses, protection against heterotypic challenge

n.t.

[148]

Rabbits

Bacillus anthracis

Protective Ag83

Protection after 2 doses

n.t.

[149]

Boosted with MVA or Human PanAd3 adults

RSV

F, NC, M

No increase and serum antibody titer and marginal increases in T cell frequencies in blood after IN prime

n.t.

[150]

HAdV5

2 doses

Macaques

Yersinia pestis

ycsF, caf1, and lcrV

Protection

No

[151]

HAdV5

1 dose

Mice

Pneumonia virus

NP, M

Weak systemic antibody and CD41 T cell responses, potent CD81 T cell responses, protection

n.t.

[152]

Vector(s)

Regimen

Species

PanAd3

1 dose

Mice

Influenza virus

Nucleoprotein (NP)
matrix protein (M) fusion protein

HAdV26, 28, 48

1 dose

Mice

Influenza virus

HAdV5

Protein boost

African green monkeys

HAdV5

1 dose

HAdV5

1 or 2 doses

PanAd3

n.t., not tested.

Vaccine target

Transgene product

Immune responses/ protection

XII. ADENOVIRUS VECTORS AS INTRANASAL VACCINES

Other researchers used replication-defective adenovirus vectors based on human or simian serotypes expressing viral or bacterial antigens in mice, guinea pigs, pigs, or nonhuman primates. The adenovirus vectors were used as single vaccine modalities or combined with DNA or, in the case of a vaccine to Mycobacterium tuberculosis, BCG primes or as booster immunizations with protein or a poxvirus vector. Most studies reported induction of antibody and T cells within the airways and protection against a subsequent challenge. Intranasal immunization elicited genital and, in some studies, intestinal antibody responses. Protective immune responses could be induced in neonatal mice [96], and responses induced in female mice were transferred and shown to be protective in their offspring [131]. A comparison of vaccines based on species C and D of adenovirus showed that vaccines from species D were less effective than those from species C upon systemic immunization but elicited equal transgene product-specific immune responses when given to the airways [146]. Some studies reported that preexisting neutralizing antibodies to the vaccine carrier had no effect on the immunogenicity of adenovirus vector vaccine given intranasally [137,142,146,151]; others disagreed [126,129]. Biodistribution studies in mice showed that adenovirus vectors upon intranasal application to mice localize to the olfactory bulb [126], but fail to disseminate to other regions of the central nervous system [127]. A clinical trial was conducted in healthy human adults with a vector based on a chimpanzee adenovirus called PanAd3 expressing an artificial fusion antigen of respiratory syncytial virus (RSV) [150]. The human volunteers had preexisting immunity to RSV due to natural infections during childhood. Vectors were given as a prime intramuscularly or intranasally. Humans were then boosted intramuscularly by a second dose of the same vector or a modified vaccinia Ankara vector. The vaccines were well tolerated, although several individuals reported a sore throat after intranasal immunization. RSV-specific neutralizing

435

antibodies in serum increased upon systemic but not intranasal prime. Circulating T cell responses increased in some individuals after the prime given through either route, but the responses were more robust and common after systemic immunization. Mucosal responses were not assessed, and it is thus not possible to draw firm conclusions about the effectiveness of intranasal immunization to elicit or boost responses within the airways. Nevertheless, lack of a robust systemic recall response upon intranasal immunization is worrisome and may predict that this route of vaccine delivery, although highly efficient in mice, is not suitable for immunization of humans. In nonhuman primates, intranasal immunization with a replication-defective HAdV5 vector expressing RSV antigens elicited a modest systemic antibody response against RSV’s fusion protein in all animals, but only a fraction responded against the viral glycoprotein [147]. Responses were more pronounced in animals that were primed with the vector given systemically. Mucosal responses were assessed after booster immunizations. Results indicate that the intranasal prime was superior at promoting responses in the airways, and it induced the highest degree of protection against intranasal RSV challenge. Similar results were obtained with a HAdV5 vaccine to Yersinia pestis, for which intranasal priming followed by a protein boost resulted in complete protection against challenge [151]. Neither of the nonhuman primate studies assessed protection after a single intranasal immunization, but rather boosted responses with a second vaccine prototype given systemically. It is thus currently impossible to conclude that intranasal adenovirus vector immunization as a single-dose vaccine modality is suited for use in primates. The apparent lack of a strong systemic immune response upon intranasal immunization of primates contrasts results in rodents and may again reflect anatomic differences of the oro-pharyngeal immune system. This may be overcome by retargeting the adenovirus vector

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

436

24. RECOMBINANT ADENOVIRUS VECTORS AS MUCOSAL VACCINES

to receptors that are more accessible than CAR within the airways. The sigma 1 protein of reovirus serotype T3 binds sialic acid and junctional adhesion molecule 1. It forms a trimer, and its structure is related to that of adenovirus fiber [153]. Adenovirus vectors with a chimeric fiber, in which the reovirus sigma I protein was fused to the C-terminus of fiber, resulted paradoxically in strongly reduced transduction rates within the airways, but improved transgene productspecific T cell responses upon intranasal immunization [154]. Further modifications of the fusion protein by increasing the length of the fusion protein resulted in improved transduction rates in the airways of mice, presumably by making the receptor-targeting domain more accessible [155]. In mice, modified vectors were shown to induce higher antibody responses in serum and in genital secretions compared to vectors with wild-type fiber. This line of research should be explored further, although its potential success would hinge not only on improved transduction rates and superior T and B cell responses, but also on genetic stability of modified vectors and vector growth characteristics, which will dictate vector yields. Overall, intranasal immunization with adenovirus vectors have been shown to prime a mucosal immune response, which, in order to become protective, may require a systemic booster immunization. Safety concerns due to transfer of the vectors into the olfactory bulb or other parts of the central nervous system may have to be addressed for some serotypes in more detail in species, such as nonhuman primates, that are more predictable for outcome in humans (For the use of adenovirus vector for HIV vaccine, see Chapter 42: Mucosal Vaccines Against HIV/SIV Infection).

XIII. IMMUNIZATIONS THROUGH THE RECTAL OR GENITAL MUCOSA Adenovirus vectors have been tested for induction of immune responses upon their

application to the genital, rectal, or colorectal mucosa of experimental animals [156
159]. We view these routes of application, which may yield basic knowledge about the mucosal immune system, as too intrusive and overall impractical for use in humans.

XIV. USE OF ADJUVANTS FOR MUCOSAL ADENOVIRUS VECTOR VACCINES Adenovirus vectors could be formulated with traditional adjuvants, or they could be designed to encode an inflammatory sequence such as the TLR-binding sequence explored in clinical trials for the oral HAdV5 vector for influenza virus [103,104]. Others demonstrated that the formulations of HAdV5 vectors in α-galactosylceramide [160], Escherichia coli heat-labile enterotoxin [161], a synthetic TLR-4 agonist [162], chitooligosaccharide-based nanoparticles with mannosylated polyethyleneiminetriethyleneglycol [163], or Ftl3 [164] further enhance mucosal transgene product-specific immune responses. We would like to caution that adenovirus vectors already induce very potent innate immune responses that cause dose-limiting toxicity, which may worsen upon the addition of adjuvants.

XV. CONCLUDING REMARKS AND FUTURE PERSPECTIVES Adenovirus vectors induce potent and sustained transgene product-specific T and B cell responses and are thus highly suited as vaccine carriers. As systemic vaccines, they are expected to be cost-effective and to provide lasting immunity after a single dose. Will they be as cheap and efficacious as mucosal vaccines? Intranasal immunization, routinely used in humans for the attenuated influenza vaccine, has yielded disappointing results in primates, which may reflect that CAR, the receptor used

IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY

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by most adenoviruses, is not expressed on the apical surface on the mucosal endothelial layer. Further studies are needed to assess whether retargeting of vectors or the use of CD46binding vectors would improve vaccine uptake into the airways and whether such vectors would meet the requirements of vaccines for mass vaccination: genetic stability, high yields during production, and efficacy in single-dose regimens. In addition, transduction of the olfactory bulb raises safety concerns that need to be addressed in more detail. The use of adenovirus vector as oral vaccines, which in many ways are more practical for mass vaccination campaigns in developing countries, appears more feasible. Results obtained in clinical trials thus far are promising. Methods to encapsidate adenovirus vectors have been developed for the live HAdV4 and HAdV7 vaccines. They may have to be adjusted to ensure thermostability of vectors and delivery of the vaccines to the ileum.

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