Novel replication-incompetent adenoviral B-group vectors: high vector stability and yield in PER.C6 cells

Abstract

Adenoviral vectors based on adenovirus type 35 (rAd35) have the advantage of low natural vector immunity and induce strong, insert-specific T-and B-cell responses, making them prime-candidate vaccine carriers. However, severe vector-genome instability of E1-deleted rAd35 vectors was observed, hampering universal use. The instability of E1-deleted rAd35 vector proved to be caused by low pIX expression induced by removal of the pIX promoter, which was located in the E1B region of B-group viruses. Reinsertion of a minimal pIX promoter resulted in stable vectors able to harbour large DNA inserts (>5 kb). In addition, it is shown that replacement of the E4-Orf6 region of Ad35 by the E4-Orf6 region of Ad5 resulted in successful propagation of an E1-deleted rAd35 vector on existing E1-complementing cell lines, such as PER.C6 cells. The ability to produce these carriers on PER.C6 contributes significantly to the scale of manufacturing of rAd35-based vaccines. Next, a stable rAd35 vaccine was generated carrying Mycobacterium tuberculosis antigens Ag85A, Ag85B and TB10.4. The antigens were fused directly, resulting in expression of a single polyprotein. This vaccine induced dose-dependent CD4+ and CD8 + T-cell responses against multiple antigens in mice. It is concluded that the described improvements to the rAd35 vector contribute significantly to the further development of rAd35 carriers for mass-vaccination programmes for diseases such as tuberculosis, AIDS and malaria.

Impact of Recombinant Adenovirus Serotype 35 Priming versus Boosting of aPlasmodium falciparumProtein: Characterization of T- and B-Cell Responses to Liver-Stage Antigen 1

“…The rAd35 vector for the LSA-1 gene (LSA-NRC sequence), optimized for expression in mammalian cells, was generated as described elsewhere (21). The recombinant LSA-1 protein (LSA-NRC sequence) was produced and purified as previously described (24).…”

“…The exceptional immunogenicity of rAd vectors is probably due to their ability to activate cells of the immune system and create a favorable cytokine and chemokine milieu for the expansion of antigenspecific immune responses (3,59). The immunogenicity of rAd, together with the availability of a highly efficient and scalable production platform (17,21), has fuelled interest in pursuing Ad vectors as a vaccine platform. The preexisting neutralizing immunity limits the use of highly prevalent serotypes, such as Ad serotype 5 (Ad5) (2,10,11,14,19,28,31,42,57,58,60); however, the development of novel vectors based on low-seroprevalence serotypes that are not influenced by the preexisting immunity toward Ad5, such as Ad35 (1,4,60), open new avenues for the use Ad vectors as vaccine vehicles.…”

Next generation: tuberculosis vaccines that elicit protective CD8+T cells

“…Since adenovirus serotype 5 is a common viral infection among people, recombinant adenovirus serotype 5-based vaccines are particularly vulnerable to failure because of pre-existing immunity. The development of rAd systems that use relatively rare adenovirus serotypes [62], or replace rAd5 coat proteins with those from rare Ad serotypes [63] are strategies currently being pursued to circumvent anti-vector immunity. Additionally, phase II trials using rAd5-based HIV vaccines will provide additional information about the extent to which pre-existing immunity negatively affects the efficacy of these vaccines.…”

Applications and challenges of multivalent recombinant vaccines

“…5,6 Extensive experience has been gathered using subunit vaccine formulation, 7 DNA, viral and bacterial vectors ( Table 1). Importantly, candidate vaccines should be safe, induce humoral and cellular immune responses against the transgene, and should provide long-lasting protection.…”

“…In the replication-defective kind, one or more gene(s) that play a role in virus replication are removed or mutated, and the resultant virus will not reproduce itself as it can only infect one cell. 6 Replicating viral vectors infect and reproduce in cells using the cell's machinery. 16,21 The new copies of the virus generated can subsequently infect other cells but because it has been manipulated it cannot cause disease.…”