Characterization of transgene expression in adenoviral vector-based HIV-1 vaccine candidates
© Takahashi et al; licensee BioMed Central Ltd. 2010
Received: 2 December 2009
Accepted: 18 February 2010
Published: 18 February 2010
Recombinant adenovirus vectors have been extensively used in gene therapy clinical studies. More recently, the capability of inducing potent cell-mediated and humoral immunity has made these vectors equally attractive candidates for prophylactic or therapeutic vaccine applications. Merck and Co., Inc., developed HIV-1 vaccine candidates based on adenovirus serotype 5 (Ad5) vectors in which the E1 gene, a critical component for adenovirus replication, was replaced by the cytomegalovirus immediate/early promoter, followed by mutated versions of the HIV-1 gag, pol or nef genes (constructs referred to as MRKAd5gag, MRKAd5pol and MRKAd5nef, respectively). Vaccine performance was evaluated in vitro in a novel assay that measures the level of transgene expression in non-permissive A549 cells. Various combinations of vectors were studied. The results indicate that the vaccine induces a dose-dependent expression of the HIV-1 transgenes in vitro. Furthermore, the gag, pol, and nef transgenes are expressed differentially in A549 cells in an MOI-dependent and formulation-dependent manner, yielding an unexpected enhancement of protein expression in trivalent vs. monovalent formulations. Our data suggest that the presence of additional virus in multivalent formulations increases individual transgene expression in A549 cells, even when the amount of DNA encoding the gene of interest remains constant. This enhancement appears to be controlled at the transcriptional level and related to both the total amount of virus and the combination of transgenes present in the formulation.
Recent clinical trials of Adenovirus-based HIV vaccines failed to demonstrate significant efficacy in protecting humans from HIV-1 infection or limiting viral load, despite strong pre-clinical immune response . Nonetheless, in preparation for clinical studies, significant development took place to characterize these vaccines. In this case, we investigated the use of non-permissive A549 cells as an in vitro model for Adenovirus type 5-based gag, pol and nef transgene expression .
The ability of an Adenovirus-based vaccine to elicit a clinical response is dependent on its ability to deliver the appropriate transgene for expression in the vaccinee; therefore, determining the levels of transgene expression of a given vector can provide an appreciation of the efficiency with which the vector has delivered the transgene, offering a measure of the vaccine's relative in vitro potency [3–5]. A549 cells were chosen specifically for their inability to support recombinant Ad5 replication , such that all transgene expression would be the result of a single round of transgene delivery and transcription/translation. Expression of each of the three transgenes in this cell line was analyzed simultaneously by SDS-PAGE and ELISA, while Reverse Transcriptase PCR (RT-qPCR) was used to quantitate mRNA.
Fold-increase in transgene expression when comparing A549 cells infected with Ad5 trivalent relative to Ad5 monovalent formulations.
Protein fold increase (95% CI)a
mRNA fold increase (95% CI)b
1.5 (± 0.3)
1.5 (± 0.2)
1.0 (± 0.1)
2.2 (± 0.2)
1.7 (± 0.1)
1.6 (± 0.1)
To explain this transgene expression enhancement in the trivalent formulation we hypothesized that either the presence of additional transgenes impacted the transcription of each given gene or that the presence of additional viral load (3-fold higher in the trivalent formulation) somehow facilitated the overall transgene expression.
To investigate whether the increase of transgene expression observed with the trivalent formulation was due to the higher amount of virus infecting the host cells or related to the simultaneous presence of one or more transgenes, we infected A549 cells with monovalent formulations of MRKAd5gag, MRKAd5pol or MRKAd5nef, supplemented either with "empty vectors" (EV, Ad5 constructs generated in the same way as the transgene-containing constructs, but missing the transgene cassette) or with a combination of EV and another monovalent vector to reach the equivalent number of virus particles that the cells were exposed to with the trivalent formulation. In this way, EV can normalize total non-replicating Ad5 virus concentration while leaving individual transgene concentration constant.
Surprisingly, ELISA analysis from cells infected with EV and either monovalent MRKAd5gag or MRKAd5nef revealed that the presence of EV in a given monovalent formulation dramatically boosted transgene expression, even exceeding the transgene expression level of the trivalent formulation (Figure 3). Whereas a 50% enhancement in transgene expression was observed in trivalent relative to monovalent formulations for gag and nef (determined by parallel line analysis on the full dose response curves), the addition of EV led to 190% and 270% increases in gag and nef transgene expression, respectively. Unlike gag and nef, no significant changes in pol transgene expression levels could be detected in the various infection combinations (Figure 3).
The data suggest that for an experimental adenovirus-based HIV-1 vaccine, transgene expression in non-permissive cells in vitro can be reproducibly modulated by the presence of additional replication-incompetent adenovirus. This modulation can occur in circumstances where another transgene-encoding adenovirus is present, as well as with the addition of adenovirus that does not contain the coding region for any transgene of interest. The mere presence of additional "empty" adenovirus particles devoid of transgene appears to enhance transgene expression. Our findings suggest these unexpected results may be worth consideration during dose and potency assay development for adenovirus-based therapeutics. Additional work is required to elucidate the mechanism of the observed transgene expression enhancement by the presence of additional adenoviral vectors. The data presented suggest that the mechanisms responsible for this enhancement are functioning prior to or during the transcription of the vector transgene. We hypothesize that the presence of additional virus results in an increase of cellular stress signals that activate transcription regulators such as MAP kinase and/or NFκB which in turn could up-regulate the CMV promoter activity driving transgene expression [12, 13].
We thank Qinjian Zhao, Shawn Zhang and Margie Geary for their contributions to the development of the gag, pol, and nef ELISAs, and Eddie Takahashi for performing MALDI-TOF analysis of pol and nef extracted SDS-PAGE bands. We also thank John Hennessey, PK Tsai, and Liman Wang for their technical input and oversight and Bob Sitrin for infrastructure and support.
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