Enhancement of capsid gene expression: preparing the human papillomavirus type 16 major structural gene L1 for DNA vaccination purposes

Abstract

Expression of the structural proteins L1 and L2 of the human papillomaviruses (HPV) is tightly regulated. As a consequence, attempts to express these prime-candidate genes for prophylactic vaccination against papillomavirus-associated diseases in mammalian cells by means of simple DNA transfections result in insufficient production of the viral antigens. Similarly, in vivo DNA vaccination using HPV L1 or L2 expression constructs produces only weak immune responses. In this study we demonstrate that transient expression of the HPV type 16 L1 and L2 proteins can be highly improved by changing the RNA coding sequence, resulting in the accumulation of significant amounts of virus-like particles in the nuclei of transfected cells. Data presented indicate that, in the case of L1, adaptation for codon usage accounts for the vast majority of the improvement in protein expression, whereas translation-independent posttranscriptional events contribute only to a minor degree. Finally, the adapted L1 genes demonstrate strongly increased immunogenicity in vivo compared to that of unmodified L1 genes.

FIG. 1

Expression constructs containing the L1 and L2 ORFs of HPV-16 with codons optimized for expression in human cells (L1h, L2h), plant cells (L1p), or with their original codons (L1ori, L2ori). In all constructs the expression of the capsid gene is driven by pCMV. To analyze the transient expressions of L1 and L2, the eukaryotic expression vector pUF3 was used (42) (a and d). This vector contains a small intron with a splice donor and splice acceptor site (SD/SA) located upstream of the respective capsid gene. (b) To analyze the influences of the various L1 genes on the expression of GFP (eGFP), bicistronic constructs in which the GFP gene was placed under the control of an IRES located downstream of the respective L1 gene were used. As a control, the L1 gene was replaced by the ecotropic retrovirus receptor gene rec1. (c) Expression construct containing L1ori in combination with the simian retrovirus CTE element cloned into the pcDNA 3.1 expression vector (Invitrogen).

FIG. 2

Adaptation for codon usage improves the expression of HPV-16 L1. (a and b) Western blot analysis of L1 expression in 293T cells upon transfection with the L1h, L1p, and L1ori expression constructs. Extracts of transfected and untransfected cells (control) were analyzed by Western blotting using the L1-specific monoclonal antibody CamVir-I. To quantitatively compare levels of L1 expression from the various constructs, different amounts of extracts were loaded: 1/10 (a) and 1:100 (b) of the L1h extract compared to the L1p and L1ori extracts. (c) L1h expression in the nuclei of transiently transfected 911 cells by indirect immunofluorescence. (d) Western blot experiment to compare the expression of L1h to the expression of L1ori constructs containing the simian retrovirus CTE element for nuclear export of L1 mRNA.

FIG. 3

Transient expression of L1 from the L1h expression construct leads to the formation of VLP in the nuclei of transfected cells. Electron micrographs of ultrathin sections of 911 cells transfected with the L1h expression construct are shown. Bar, 0:2 μm. Cy, cytosol; NM, nuclear membrane; Nu, nucleus.

FIG. 4

Influence of various HPV-16 L1 ORFs on the expression of a downstream-located GFP gene in a bicistronic expression construct. Cells were transfected with expression constructs containing genes encoding L1h, L1p, and L1ori, or a non-L1 gene (the ecotropic retrovirus receptor gene rec1) upstream of an IRES-GFP cassette. Expression of GFP was analyzed by FACS analysis (a) and Western blotting (b). Note that the total fluorescence of the rec1-GFP-transfected cells was set to 100%. (c) Western blot analysis of L1 expression levels in the same extracts as for panel b.

FIG. 5

Expression of codon-optimized HPV-16 L2. 293T cells were transfected with expression constructs containing HPV-16 L2ori, HPV-11 L2ori or HPV-16 L2h. The L2 protein was subsequently detected by Western blotting in extracts of transfected cells by use of a polyclonal rabbit antiserum specific for HPV-16 and HPV-11 L2 (a) or by indirect immunofluorescence of transfected cells (b).

FIG. 6

Induction of L1-specific antibodies by DNA immunization. Fifteen mice falling into four groups were immunized by injection of plasmid DNA. Anti-L1-specific antibodies were measured by an HPV-16 VLP-specific ELISA. ▪, five mice immunized with the expression plasmid containing the L1h gene; ○, five mice immunized with the plasmid containing the L1ori gene. The control group consisted of four mice immunized with an expression vector containing a non-L1 gene (VP22-E7) (▴) and one nonimmunized mouse (⧫).