Adenoviral Vector for Vaccine Development

Adenoviral vectors were initially investigated for years as vehicles for gene therapy. However, attempts to replace missing or faulty genes by adenoviral vector gene transfer were largely unsuccessful in experimental animals and human volunteers most likely because of innate and adaptive immune responses induced by the adenoviral antigens. Currently, adenovirus is known to interact with several different extracellular, intracellular, and membrane-bound innate immune sensing systems (Hartman et al. 2008). Adenovirus expresses pathogen-associated molecular patterns (PAMPs), which bind to pathogen recognition receptors (PRRs) on host cells, including those of the innate immune system, in order to produce the proinflammatory chemokines and cytokines and to differentiate the immature dendritic cells into professional antigen-presenting cells (Hartman et al. 2008; Medzhitov and Janeway 2000). Adaptive immune responses are directed to both early and late viral antigens of adenovirus. Adenovirus-neutralizing antibodies induced after adenoviral infection or upon adenoviral vector administration are primarily directed against the surface loops of the viral hexon (Wohlfart 1988), even though antibodies targeted to the penton base or the fiber can also neutralize adenovirus (Hong et al. 2003). In addition, a highly conserved human CD4+ T-cell epitope has been identified within adenovirus capsid protein hexon (Olive et al. 2002).

So far, adenoviruses are among the most heavily exploited vectors for vaccine development. Adenovirus can grow to high titer in vitro with physical and genetic stability. In addition, adenovirus infects both dividing and non-dividing cells without integration in the host genome and yields high levels of antigen expression. More importantly, adenovirus infected dendritic cells upregulate costimulatory molecules and induce cytokine and chemokine responses, thus effectively presenting antigens to the immune system and eliciting potent antigen-specific immune responses (Banchereau and Steinman 1998; Morelli et al. 2000; Molinier-Frenkel et al. 2003; Philpott et al. 2004).

Currently, non-replicating adenoviral vaccine vectors are created by the deletion of E1 region genes, which are essential for replication. Such vaccine vectors generally also have the non-essential E3 region deleted in order to provide more space for the completed expression cassette for antigen, including exogenous promoter, a transgene for antigen, poly (A) signal, etc. It has been shown that non-human primates intramuscularly immunized with non-replicating adenoviral vaccine vector expressing SIV Gag gene were protected against experimental infection with a chimeric SIV/HIV virus better than those intramuscularly immunized with plasmid DNA or the modified vaccinia Ankara (MVA) recombinant encoding the same antigen (Shiver et al. 2002). The prime and boost approaches, especially priming with improved plasmid DNA vaccines to focus the immune responses on the foreign antigen followed by a boost with an adenoviral vaccine vector expressing the same antigen, were shown to enhance the efficacy of protection (Casimiro et al. 2005; Mattapallil et al. 2006). In addition, non-replicating adenoviral vaccine vector encoding rabies virus glycoprotein can induce protective neutralizing antibody titers against rabies virus rapidly after only a single application. The efficacy of this non-replicating adenoviral vaccine was far superior to that of a well-characterized vaccinia rabies glycoprotein recombinant (Xiang et al. 1996). A recent study suggested that non-replicating adenoviral vaccine vectors were transcriptionally active at low levels for long periods of time after intramuscular immunization in mice (Tatsis et al. 2007). Continuously produced small amounts of antigen help to maintain fully active antigen-specific effector CD8+ T-cells, which can further differentiate into central memory cells. The long-term persistence of adenoviral vaccine vectors might be highly beneficial against pathogens, which require both fully activated effector CD8+ T-cells for immediate control of pathogen-infected cells and central memory CD8+ T-cells for elimination of pathogen-infected cells that escaped the initial wave of effector CD8+ T-cells.

Non-replicating adenovirus vectors with deletion of E1 and E4 regions have been explored mainly for gene therapy (Mizuguchi and Kay 1999). E4-deleted adenovirus vectors produce fewer late viral gene products than only E1-deleted vectors. Therefore, it will be interesting to test whether E1- and E4-deleted adeno-virus vaccine vectors can indeed induce less vector-specific and more focused antigen-specific immune responses than only E1-deleted vectors.

Replicating adenoviral vaccine vectors contain the deletion only in the E3 region. Therefore, replicating adenoviral vaccine vectors have a more limited cloning capacity for transgenes. The main scientific advantage of replicating adenoviral vaccine vectors is that they can elicit more potent immune responses, including innate immunity, as well as humoral, cellular, and mucosal immune responses. The wild-type adenoviral vaccine vectors were proven to be safe after they were used for over 26 years in the US military (Robert-Guroff 2007). In replicating adenoviral vaccine vectors, expression of the encoded antigen is incorporated into the viral replication cycle so that lower immunization doses can achieve longer and higher expression levels of antigen in vivo than non-replicating adenoviral vaccine vectors. In addition, adenoviral replication in vivo can stimulate production of proinflam-matory cytokines, further augmenting immune responses. Apoptotic cells arising from adenoviral replication can provide DC with exogenous antigens for initiation of T-cell responses through cross-presentation (Fronteneau et al. 2002). Although replicating adenoviral vaccine vectors may compete with transgenes for induction of immune responses, strong immune responses to adenoviral antigens may paradoxically enhance immunity to transgene-encoded antigen via CD8-T-cell-mediated autocrine help (Truckenmiller and Norbury 2004), whereby CD8+ T-cells can provide help for other responding CD8+ T-cells if present in sufficient numbers (Wang et al. 2001). A comparative study in chimpanzees after immunization with either replicating or non-replicating adenoviral vaccine vector encoding the same antigen demonstrated both the greater induction of cellular immune responses and the ability to prime more potent antibody responses by the replicating adenoviral vaccine vector (Peng et al. 2005).

Virus-specific neutralizing antibodies even at moderate titers can significantly reduce uptake of the adenoviral vaccine vectors by cells, including antigen-presenting cells, after intramuscular immunization, which in turn impacts the transgene product-specific immune responses (Fitzgerald et al. 2003). Pre-existing immunity in general human population due to natural infections of the common serotypes of human adenovirus, such as serotype 5 (Ad5) and serotype 2 (Ad2), results in sustained virus-neutralizing antibody titers. Therefore, a way to circumvent the impact of pre-existing adenovirus-specific immunity in humans is essential for the success of adenoviral vaccine vectors in the clinical setting. Simple dose escalation is unlikely to be a realistic option because of the associated toxicity. Studies in Ad5-preexposed nonhuman primates showed that a 1,000-fold increase in dose is needed to achieve frequencies of transgene product-specific CD8+ T-cells comparable to those obtained in animals that have not been pre-exposed (Casimiro et al. 2003). Currently, alternative serotypes and strains of adenovirus from different species are being developed as vaccine carriers. Adenovirus of rare serotype such as serotype 11 (Ad11), serotype 26 (Ad26), serotype 35 (Ad35), and serotype 49 (Ad49) can be used in sequential prime-boost regimens of intramuscular immunization in order to avoid or lessen the impact of pre-existing immunity as well as cross-reactivity between adenovirus serotypes (Thorner et al. 2006). In addition, vaccine vectors based on the non-human adenoviruses of chimpanzee origin (Farina et al. 2001; Tatsis et al. 2006), and the engineered chimeric vectors in which the hypervariable regions of the hexon protein of Ad5 are replaced with corresponding regions of a rare adenovirus serotype 48 (Ad48) (Roberts et al. 2006), have been shown to effectively overcome the pre-existing anti-vector immunity. Simian E1-deleted adenoviral vectors, such as AdPan9, AdPan7, and AdPan6, showed comparable yields upon propagation on HEK 293 cells, indicating that they can grow in the presence of E1 of Ad5. As the sequences flanking the E1 region show limited homology between different serotypes of adenoviruses, the risk of contamination of E1-deleted simian adenoviral vaccine vectors with replicating adenovirus due to the homologous recombination of simian vaccine vectors with Ad5 E1 of the packaging cell line is virtually absent. Overall, these alternative adenoviral vaccine vectors provide the flexibility in primer-boost strategy and focus the immune response on the inserted antigen while avoiding anti-vector immunity induced by prior natural infection or immunizations. However, these alternative adenoviral vaccine vectors will require extensive safety testing and their relative immunogenicity in comparison to Ad5 will need to be clearly established.

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