Determinación de los parámetros de control de calidad de la droga cruda

The Ad5 vaccine vector system has not, to date, yielded a successful vaccine platform for use in the clinic. However, their use as potential vaccine vectors has not subsided. A large variety of replication-deficient vaccine vectors exist with alternative gene deletions which reduces potential side effects associated with replication-competent vectors. This characteristic also ensures that they can be used to immunise immunocompromised individuals without the concern of overwhelming the immune system. The molecular cloning techniques have produced commercially available kits, such as the pAdEasy (Strategene) and pEntry (Invitrogen) systems. These kits allow the rapid generation and isolation of research grade Ad5 vectored vaccines to allow the rapid investigation of potential candidates.

Ads also have a broad cell tropism (Nanda et al., 2005). Ad5 in particular uses the Coxsackie adenovirus receptor (CAR) as its primary cell receptor which is expressed on many cell types, including myoblasts, hepatocytes and epithelial and endothelial cells (Tatsis & Ertl, 2004). In the airway, CAR is not expressed but Ad5 is able to utilise an alternative isoform to infect these cells (Excoffon et al., 2010). This characteristic ensures a large number of cells can be transduced, maximising

transgene antigen exposure to the immune system. Therefore, the route of immunisation can be chosen to best stimulate the type of immune response needed to provide protection against a pathogen. In addition, Ad5 can infect dividing and non- dividing cells (Tatsis & Ertl, 2004). This also ensures that transgene expression can be supported in a large number of cells.

Standard Ad5 vectors are amenable to large scale growth for commercial production as they can grow to high titres in a stable manner. The generation of stable replication-complementing mammalian cell lines, such as HEK293 cells, allows the replication-deficient viruses to be propagated in tissue culture. This reduces the potential for contamination with other viruses or helper viruses, which could lead to recombination and reversion to a replication-competent virus. Furthermore, Ad5 does not routinely integrate into the host cell chromosomes, improving the safety of the vector (Xu et al., 2009).

Furthermore, Ads naturally stimulate a strong immune response which is biased towards a Th1 response both via systemic and mucosal immunisation routes as

discussed below in Section 1.6.2 (Santra, 2005). Upon entry to a host cell, innate immune responses are triggered, which in turn stimulate the adaptive response. Ads stimulate high avidity and high titre neutralising antibodies against encoded transgenes, in addition to a strong CD8+ T-cell response to both immunodominant and non-immunodominant epitopes (Barefoot et al., 2008, Santra, 2005, Tatsis & Ertl, 2004). This can greatly enhance the potency of the vaccine as, if an increased array of epitopes is recognised by the immune system, it decreases the chance of virus escape (Santra, 2005). Although this factor is not important for vaccines against more stable viruses, those with a high mutagenic rate, such as HIV and influenza, are therefore less likely to overcome the immune system and result in disease.

Pre-existing immunity towards Ad5 has been shown to have a negative impact on the immune response towards the transgene, reducing transgene expression from months to weeks (Zaiss, 2009). In addition, the immunogenicity of Ad5 can affect the efficacy of a vaccine if multiple doses are required, as these generate strong anti-Ad5 immune responses (Santra, 2005). Neutralising antibody typically targets the hexon, fibre and penton proteins and the cellular response is often directed to the E1a, E1b,

E2a and hexon proteins (Gahery-Segard et al., 1998, Thacker et al., 2009, Yang et al., 1995). Serological studies have indicated a high prevalence of anti-Ad5 immunity in the human population, which may hinder vaccine efficacy in these populations (Mast et al., 2009). However, some studies have indicated anti-Ad5 immunity may not impede an immune response towards a vaccine transgene (Casimiro et al., 2003). One particular study using a bovine Ad vector demonstrated that seropositive animals were able to mount an effective immune response against the vaccine transgene in spite of pre-existing immunity against the vector (Babiuk & Tikoo, 2000). Another group investigating this effect in humans also confirmed this finding (Gahery-Segard et al., 1998). Therefore, other groups suggested Ad5 could be used as part of a heterologous (rather than as a homologous) immunisation regime to minimise anti-Ad immunity issues (Natuk, 1994, Reyes-Sandoval, 2010, Santra, 2005, Shiver et al., 2002). Heterologous regimes involve immunising individuals with several different types of vaccine preparation such as live recombinant, DNA and subunit, whereas homologous regimes involve immunisation with the same vaccine preparation. In response to this issue, alternative Ad serotypes are currently being investigated as suitable vaccine vectors. Serological studies have suggested that Ad25, Ad11, Ad35 and Ad3 could be used as potential vectors, however, these have been shown to be less immunogenic than Ad5, in terms of the transgene response elicited (Abbink, 2007, Barouch et al., 2004, Lemckert et al., 2005, Li et al., 2009, Mast et al., 2009). Furthermore, these constructs are also less immunogenic than Ad5 when used in heterologous and homologous immunisation strategies (Santra et al., 2009). One solution is to replace the fibre knob of Ad5, the receptor attachment site, with that of another serotype, such as Ad35; this has been shown to retain immunogenicity but circumvent pre-existing immunity (Nanda et al., 2005). In addition, animal Ads particularly chimpanzee Ad serotypes such as, ChAd7, ChAd68 and ChAd1/5 are under consideration as potential vectors because humans do not have pre-existing immunity towards them (Dudareva et al., 2009, McCoy et al., 2007, Peruzzi et al., 2009).