Characterization of transgene expression in adenoviral vector-based HIV-1 vaccine candidates

Marie-Noëlle Takahashi,Judith A Rolling,Katherine E Owen

doi:10.1186/1743-422x-7-39

Marie-Noëlle Takahashi, Judith A Rolling + Show 1 more

Open Access

https://doi.org/10.1186/1743-422x-7-39

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Journal: Virology journal	Publication Date: Feb 18, 2010
Citations: 14	License type: cc-by

Affiliation: United States Military Academy

Abstract

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.

Highlights

Recombinant adenovirus vectors have been extensively used in gene therapy clinical studies
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 [2]
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; 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 [3,4,5]

Summary

Introduction

Recombinant adenovirus vectors have been extensively used in gene therapy clinical studies. Comparison of transgene expression levels in A549 cells infected with monovalent Ad5 vectors made using a direct detection approach with SDS-PAGE followed by silver staining: analysis of infected cell lysates revealed that the nef protein was more intense than the pol protein (Figure 1C), suggesting that ELISA and RTqPCR results were not an artifact of the detectors.

Results

Conclusion